Polypeptides involved in immune response

ABSTRACT

Polypeptides which comprise a receptor-ligand pair involved in T-cell activation are disclosed. Nucleic acid molecules encoding the polypeptides, and vectors and host cells for expressing the polypeptides are also disclosed. The polypeptides, or agonists and antagonists thereof, are used to treat T-cell mediated disorders.

This application is a divisional of U.S. Ser. No. 11/359,254, filed Feb.21, 2006 now U.S. Pat. No. 7,708,993, now allowed, which is a divisionalof U.S. Ser. No. 09/728,420, filed Nov. 28, 2000, now abandoned, whichis a continuation-in-part of international application Serial No.PCT/US00/01871, with an international filing date of Jan. 27, 2000, nowissued as U.S. Pat. No. 7,435,796, which is a continuation-in-part ofapplication Ser. No. 09/264,527, filed Mar. 8, 1999, now abandoned,which is a continuation-in-part of application Ser. No. 09/244,448,filed Feb. 3, 1999, now abandoned, which are hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to polypeptides that are involved inT-lymphocyte activation. Specifically, the invention relates toT-lymphocyte costimulatory polypeptides, the nucleic acids encoding thepolypeptides, expression vectors and host cells for production of thepolypeptides, and compositions and methods for the treatment of diseasesrelated to immunosuppression and immune activation.

BACKGROUND OF THE INVENTION

For the generation of a proper T-lymphocyte (T-cell) immune response,two signals must be provided to the T-cell by antigen presenting cells(APC). First, antigen must be presented to the T-cell receptor (TCR) viaa major histocompatibility complex (MHC), in an event that determinesspecificity. Second, an antigen-independent, costimulatory signal mustbe delivered by engagement of members of the B7 family on the APC withthe CD28 protein on T-cells. A productive immune response leads toproliferation, differentiation, clonal expansion, and effect orfunction. In the absence of the second, costimulatory signal, T-cellsundergo a state of long-lasting antigen-specific unresponsiveness,termed anergy.

T-cells initiate the immune response, mediate antigen-specific effectorfunctions, and regulate the activity of other leukocytes by secretingcytokines. The T-cell receptor (TCR) distinguishes the T-cell from otherlymphocytes and can bind antigen only when it is presented by the APCwithin the context of a MHC. The functional activity of a particularT-cell can be correlated with the expression of membrane antigens, suchas CD4 and CD8. For instance, CD4+ T-cells generally function as Thelper cells (T_(H)) and are MHC class II restricted, whereas CD8+ cellsgenerally function as cytotoxic T-cells (T_(C)) and are MHC class Irestricted.

Potent T-cell costimulatory polypeptides which has been previouslyidentified include polypeptides termed B7.1 (Freeman et al. J.Immunology 143, 2714-2722 (1989), Freeman et al. Jour. Expt. Med. 174,625-31 (1991)) and B7.2 (Freeman et al. Science 262, 909-911 (1993), andFreeman et al. Jour. Expt. Med. 178, 2185-2192 (1993)), (or CD80 andCD86, respectively). These polypeptides are either inducibly orconstitutively expressed on various APCs and are membrane-bound ligandsfor CD28 and CTLA-4, respectively, on T-cells. CD28 (Aruffo and SeedProc. Natl. Acad. Sci. 84, 8573-8577 (1987) and Gross et al. J. Immun.144, 3201-3210 (1990)) is expressed on resting T-cells and mediates apositive costimulatory signal. CTLA-4 (Brunet et al. Nature 328, 267-270(1987) and Dariavach et al. Eur. Jour. Immun. 18, 1901-1905 (1988))expression is induced upon T-cell activation and negatively regulatesthe CD28 signal, due to its higher binding affinity for B7.1 and B7.2.Mice without the CTLA-4 gene exhibit dramatically high levels ofT-cells, since the switch off mechanism for the proliferation signal isimpaired in the absence of CTLA-4. This phenotype clearly demonstratesthe major inhibitory effect that the CTLA-4 costimulatory protein has onT-cell proliferation. Mice lacking CD28 or B7.1 or B7.2 have a lesssevere phenotype, indicating that alternate pathways for T-cellcostimulation may exist.

There has been considerable interest in the CD28/CTLA-4 pathway as meansfor regulating T-cell activation and proliferation. A chimeric proteincontaining the extracellular portion of CTLA-4 fused to human Fc hasstrong immunosuppressive effects and has been studied in a variety ofclinical settings. Antibodies to B7.1 and B7.2 proteins have also beenevaluated for similar indications in the area of immunosuppression.Anti-CTLA-4 antibodies have shown utility in promoting T-cellactivation. In addition, B7.1 and B7.2 gene therapy has shown greatpromise in the area of cancer immunotherapy.

Thus far, CD28, CTLA-4, B7.1 and B7.2 are involved in a single T-cellcostimulatory pathway. Given the capability of modulating an immuneresponse by regulating T-cell costimulation, it would be desirable toidentify other members of the same or a separate T-cell costimulatorypathway which may have advantageous properties in regulating host T-cellfunction and immune response.

Accordingly, it is an object of the invention to provide novelpolypeptides for stimulation of T-cell activity and/or proliferation. Itis a further object of the invention to use the novel polypeptides forthe prevention and treatment of T-cell mediated immune disorders.

SUMMARY OF THE INVENTION

Surprisingly, two novel polypeptides of a T-cell costimulatory pathwayhave been identified. The polypeptides represent a ligand-receptor pairin a unique costimulatory pathway which appears to be distinct from thepathway consisting of previously described proteins CD28, CTLA-4, B7.1,and B7.2. The polypeptides are referred to as CD28 related protein-1, orCRP1, and B7 related protein-1, or B7RP1.

The invention provides for nucleic acid molecules encoding CRP1 andB7RP1 polypeptides and related polypeptides. An isolated nucleic acidmolecule of the invention comprises a nucleotide sequence selected fromthe group consisting of:

a) the nucleotide sequence as set forth in FIG. 1A (SEQ ID NO:1);

b) the nucleotide sequence encoding the polypeptide from residues 1-200or from residues 21-200 as set forth in FIG. 1A (SEQ ID NO:1);

c) a nucleotide sequence encoding a polypeptide that is at least about70 percent identical to the polypeptide as set forth in FIG. 1A (SEQ IDNO: 1);

d) a naturally occurring allelic variant or alternate splice variant ofany of (a), (b) or (c);

e) a nucleotide sequence complementary to any of (a), (b) or (c);

f) a nucleotide sequence of (b), (c) or (d) encoding a polypeptidefragment of at least about 25, 50, 75, 100, or greater than 100 aminoacid residues;

g) a nucleotide sequence of (a), (b) or (c) comprising a fragment of atleast about 10, 15, 20, 25, 50, 75, 100, or greater than 100nucleotides; and

h) a nucleotide sequence which hybridizes under stringent conditions toany of (a)-(g).

Also provided by the invention is an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

a) the nucleotide sequence as set forth in FIG. 2A (SEQ ID NO: 6) orFIG. 3A (SEQ ID NO:11) or FIG. 12A (SEQ ID NO: 16);

b) the nucleotide sequence encoding the polypeptide as set forth in FIG.2A (SEQ ID NO: 6) from residues 1-322 or from residues 47-322 or as setforth in FIG. 3A (SEQ ID NO:11) from residues 1-288 or from residues19-288, 20-288, 21-288, 22-288, 24-288, or 28-288; or as set forth inFIG. 12A from residues 1-302 or from residues 19-302, 20-302, 21-302,22-302, 24-302 or 28-302;

c) a nucleotide sequence encoding a polypeptide that is at least about70 percent identical to the polypeptide as set forth in FIG. 2A (SEQ IDNO: 6) or FIG. 3A (SEQ ID NO: 11) or FIG. 12A (SEQ ID NO: 16);

d) a naturally occurring allelic variant or alternate splice variant ofany of (a), (b) or (c);

e) a nucleotide sequence complementary to any of (a), (b) or (c);

f) a nucleotide sequence of (b), (c) or (d) encoding a polypeptidefragment of at least about 25, 50, 75, 100, or greater than 100 aminoacid residues;

g) a nucleotide sequence of (a), (b) or (c) comprising a fragment of atleast about 10, 15, 20, 25, 50, 75, 100, or greater than 100nucleotides; and

h) a nucleotide sequence which hybridizes under stringent conditions toany of (a)-(g).

The subject matter of the invention also relates to CRP1 and B7RP1polypeptides and related polypeptides. The invention provides for anisolated polypeptide comprising the amino acid sequence selected fromthe group consisting of:

a) the amino acid sequence as set forth in FIG. 1A (SEQ ID NO:2);

b) the mature amino acid sequence as set forth in FIG. 1A (SEQ ID NO:2)comprising a mature amino terminus at residue 21;

c) a fragment of the amino acid sequence set forth in FIG. 1A (SEQ IDNO: 2) comprising at least about 25, 50, 75, 100, or greater than 100amino acid residues;

d) an ortholog of (a), (b) or (c); and

e) an allelic variant or alternative splice variant of (a), (b) or (d).

Also in accordance with the invention is an isolated polypeptidecomprising the amino acid sequence selected from the group consistingof:

a) the amino acid sequence as set forth in FIG. 2A (SEQ ID NO: 7) orFIG. 3A (SEQ ID NO:12) or FIG. 12A (SEQ ID NO: 17);

b) the mature amino acid sequence as set forth in FIG. 2A (SEQ ID NO: 7)comprising a mature amino terminus at residues 47 or FIG. 3A (SEQ IDNO:12) comprising a mature amino terminus at any of residues 19, 20, 21,22, 24 or 28 or FIG. 12A (SEQ ID NO: 17) comprising a mature aminoterminus at any of residues 19, 20, 21, 22, 24, or 28;

c) a fragment of the amino acid sequence set forth in FIG. 2A (SEQ IDNO: 7) or FIG. 3A (SEQ ID NO: 12) or FIG. 12A (SEQ ID NO: 17) comprisingat least about 25, 50, 75, 100, or greater than 100 amino acid residues;

d) an ortholog of (a), (b) or (c); and

e) an allelic variant or alternative splice variant of (a), (b), (c) or(d).

Also encompassed by the invention are expression vectors and host cellsfor production of the polypeptides, antibodies which bind to CRP1 andB7RP1 polypeptides and to related polypeptides, and assays for detectingbinding of B7RP1 and B7RP1-related polypeptides to CRP1 and CRP1-relatedpolypeptides. Pharmaceutical compositions comprising CRP1 orCRP1-related polypeptides and B7RP1 or B7RP1-related polypeptides arealso encompassed by the invention. Methods for identifying compoundsthat interact with CRP1 or B7RP1 are also provided as are assays fordetermining whether such compounds are agonists or antagonists of CRP1and B7RP1 activity.

CRP1 and B7RP1 polypeptides are involved in T-cell costimulation andproliferation. CRP1 and B7RP1 polypeptides, selective binding agentsthereof, and agonists and antagonists thereof, may be useful for thediagnosis, prevention and treatment of diseases related to the controlof T-cell responses.

CRP1 and B7RP1 polypeptides, selective binding agents thereof, andagonists and antagonists thereof, may be useful for the diagnosis,prevention and treatment of immune disorders, either for stimulatinginsufficient immune response or reducing or inhibiting an exaggerated orinappropriate immune response. The immune disorder may be mediateddirectly or indirectly by T-cells.

The invention provides for treating, preventing, or ameliorating aT-cell mediated disorder comprising administering to an animal a CRP1 orB7RP1 polypeptide. The invention also provides for a method ofdiagnosing a T-cell mediated disorder or a susceptibility to a T-cellmediated disorder in an animal comprising determining the presence oramount of expression of a CRP1 or B7RP1 polypeptide; and diagnosing aT-cell mediated disorder or a susceptibility to a T-cell mediateddisorder based on the presence or amount of expression of thepolypeptide. Typically, a T-cell mediated disorder is an immune disorderwhich may be mediated directly or indirectly by T-cells. The animal ispreferably a mammal and more preferably a human The invention alsoprovides for a method of identifying a test molecule which binds to aCRP1 or B7RP1 polypeptide comprising contacting the polypeptide with atest compound and determining the extend of binding of the polypeptideto the test compound. The method may be used to identify agonists andantagonists of CRP1 and/or B7RP1 polypeptide.

Antagonists of CRP1 and/or B7RP1 polypeptides may be used asimmunosuppressive agents for many indications, including autoimmunedisorders (such as rheumatoid arthritis, psoriasis, multiple sclerosis,diabetes, and systemic lupus erythematosus), toxic shock syndrome, bonemarrow and organ transplantation, inflammatory bowel disease,allosensitization due to blood transfusions, and the treatment of graftvs. host disease. In addition, antagonists may be used as inhibitoryagents for T-cell dependent B-cell mediated indications including asthmaand allergy, and antibody mediated autoimmunity. Agonists of the CRP1and/or B7RP1 polypeptides may be useful in, but not restricted to,T-cell activation for tumor surveillance and removal.

An antagonist of the invention includes an antibody, or fragmentthereof, which is reactive with or binds to B7RP1 or to an extracellulardomain of B7RP1 wherein the antibody reduces or eliminates the bindingto B7RP1 to CRP1. In one embodiment, the antibody binds selectively tohuman B7RP1 or to an extracellular domain thereof. The antibody orfragment thereof which is an antagonist inhibits partially or completelythe immune costimulatory activity of B7RP1. In a preferred embodiment,the antibody is a monoclonal antibody and may be murine, human, chimericor humanized.

The invention further provides for a method of regulating theinteraction of B7RP1 with CRP1 comprising administering to an animal aselective binding agent of CRP1 or a selective binding agent of B7RP1 orboth. In one embodiment, the selective binding agent is an antibodywhich binds to B7RP1 and reduces or eliminates the binding to B7RP1 toCRP1. The invention also provides a method of regulating immunecostimulation mediated by B7RP1 comprising administering to an animal aselective binding agent of B7RP1. The selective binding agent ispreferably an antibody which binds to B7RP1 and partially or completelyinhibits immune costimulation mediated by B7RP1.

The invention also provides for a method of regulating T-cell activationor proliferation in an animal comprising administering to the animal anucleic acid molecule encoding a CRP1 or B7RP1 polypeptide. For example,a nucleic acid molecule encoding a B7RP1 polypeptide may be used in genetherapy to enhance T-cell activation in response to various tumors.

Also encompassed by the invention is a transgenic non-human mammalcomprising a nucleic acid molecule encoding a CRP1 or B7RP1 polypeptide.The CRP1 or B7RP1 nucleic acids are introduced into the mammal in amanner that allows expression and increased circulation levels of CRP1or B7RP1 polypeptides. The transgenic non-human mammal is preferably arodent, and more preferably a mouse or a rat.

A method for stimulating or enhancing an immune response comprisingadministering B7RP1 or CRP1, or a B7RP1 agonist or CRP1 agonist is alsoprovided. Optionally, an immune response may be stimulated or enhancedby further administering one or more other immune stimulating molecules,such as a CD28 agonist, a CTLA4 antagonist, or molecules such as B7.1and/or B7.2.

Also provided by the invention is a method of regulating IgE productioncomprising administering B7RP1 or CRP1, or a combination thereof, aB7RP1 agonist or a CRP1 agonist, or a combination thereof, or a B7RP1antagonist or a CRP1 antagonist, or a combination thereof. For example,administration of B7RP1 or a combination of B7RP1 and a CRP1 agonistwould increase IgE production and would be useful for stimulating aninsufficient IgE-mediated immune response. Administration of a B7RP1antagonist or a CRP1 antagonist, or a combination thereof, woulddecrease IgE production and would be useful for inhibiting anexaggerated or inappropriate IgE-mediated immune response. In oneembodiment, IgE production is partially or completely inhibited byadministration of a B7RP1 antagonist, or a CRP1 antagonist, or acombination thereof. In another embodiment, the invention provides for amethod of preventing and/or treating an IgE-mediated disorder comprisingadministering a B7RP1 antagonist, or a CRP1 antagonist, or a combinationthereof. IgE-mediated disorders include those characterized by excessiveIgE production, such as asthma, allergic disorders, hypersensitivity andsinus inflammation.

DESCRIPTION OF FIGURES

FIG. 1. A) DNA and amino acid sequence murine CRP1 (mCRP1) (SEQ ID NO:1). Predicted signal sequence of CRP1 is underlined at theamino-terminus and the experimentally determined pro-peptide cleavagesite is indicated by an asterisk. Predicted transmembrane sequence isunderlined toward the carboxy-terminus. B) Amino acid alignment ofmurine CRP1 protein sequence (mCRP1) (SEQ ID NO: 2) with murine CD28(mCD28) (SEQ ID NO: 4).

FIG. 2. A) DNA and amino acid sequence of murine B7RP1 (mB7RP1) (SEQ IDNO: 6). Predicted signal sequence of B7RP1 is underlined at theamino-terminus and the experimentally determined pro-peptide cleavagesite is indicated by an asterisk. Predicted transmembrane sequence isunderlined toward the carboxy-terminus. B) Amino acid alignment of B7RP1protein sequence (mB7RP1) (SEQ ID NO: 7) with murine CD80 (mCD80) (SEQID NO: 9).

FIG. 3. A) Structure and sequence of the protein coding region of theputative human B7RP1 (hB7RP1) (SEQ ID NO: 11). Predicted signal sequenceof hB7RP1 is underlined at the amino-terminus. Predicted signal peptidecleavage sites are marked by asterisks. Predicted transmembrane sequenceis underlined toward the carboxy-terminus. B) Amino acid alignment ofthe putative mature hB7RP1 protein (SEQ ID NO: 13) with the maturemurine B7RP1 (mB7RP1) protein (SEQ ID NO: 14).

FIG. 4. A) Expression of soluble CRP1-Fc fusion protein from 293T-cellstransfected with the pcDNA3/CRP1-Fc. Normalized volumes of cell lysateor conditioned medium were loaded and separated on a 10% PAGE gel asindicated. Western analysis of cell lysate and cell media supernatantfor expression of cell-associated (cell lysate) and secreted (media) Fcfusion proteins. Primary antibody was Goat-anti human Fc antibody(Pierce Chemical Company, Rockford, Ill.). B) Expression of solubleB7RP1-Fc fusion protein from 293T-cells transfected with thepcDNA3/B7RP1-Fc. 20 μl of normalized cell lysate or media supernatantwere loaded and separated on a 10% PAGE gel. Western analysis wasconducted as in (A).

FIG. 5. Interaction of CRP1-Fc and B7RP1-Fc fusion proteins withmembrane-bound proteins expressed in COS-7 cells. COS-7 cellstransiently transfected with pcDNA3/CRP1, pcDNA3/B7RP1, or pcDNA3 vectoralone. CHO D-cells were transfected with psDRα/hCD28 and stablyexpressed human CD28 (hCD28). Cells expressing membrane-bound CRP1,B7RP1, or hCD28, are represented in rows as indicated at the left sideof the panel. Fc fusion proteins were incubated with the plate-boundcells in columns as indicated at the top of the panel. After incubation,cells were washed, and bound Fc fusion proteins were detected using ananti-human Fc antibody and ACAS (Adherent Cell Analysis and Sorting;ACAS Ultima, Meridian Instruments, Inc., Okemos, Mich.) analysis.

FIG. 6. FACS (Fluorescence-Activated Cell Sorter) analysis of expressionof the receptor for B7RP1 (putatively, CRP1) on activated CD4+ and CD8+T-cells. Mouse splenocytes were activated with PMA and ionomycin for 12hours. B7RP1-Fc fusion protein, control Fc protein (Mock-Fc), or PBS (nostain), were incubated with the cells, washed, and subsequentlyincubated with goat-anti-human Fc-FITC conjugated antibody (GaHuFc-FITC)as indicated at the bottom of each panel. Cell marker antibodies (forT-cell markers CD4 and CD8) PE conjugated, or isotype control antibody(rat isotype) PE conjugated, or PBS (no stain), were added as indicatedat the left side of each individual panel.

FIG. 7. FACS (Fluorescence-Activated Cell Sorter) analysis of theexpression of B7RP1 on B-cells. Fluorocytometric analysis of theexpression of the ligand for CRP1 (presumably, B7RP1) on mousesplenocytes. CRP1-Fc fusion protein, control Fc protein (Mock-Fc), orPBS (no stain), were incubated with the cells, washed, and subsequentlyincubated with goat-anti-human Fc-FITC conjugated antibody (GaHuFc-FITC)as indicated at the bottom of each panel. PE conjugated cell markerantibody to CD45R (CD45R is a B-cell marker) or isotype control antibody(rat isotype), or PBS (no stain), were added as indicated at the leftside of each individual panel.

FIG. 8. FACS analysis of the expression of mCRP1 ligand on peritonealmacrophages. Peritoneal cells were first distinguished in subsets on theground of their light scattering properties (panel A). Macrophages wereidentified in region 5 (R5) because of their ability to strongly scatterlight forward (FSC) sideways (SSC) and because of their positivestaining for the F4/80 antigen, a marker for macrophages (panel B).Macrophages in region 6 (R6) were singled out on the basis of their lessintense staining for the F4/80 antigen and found to be stained by theCRP1-Fc fusion protein (presumably because of their expression ofB7RP1).

FIG. 9. Inhibition of T-cell proliferation using a B7RP1-Fc fusionprotein. T-cells from mouse splenocytes were activated by increasingconcentrations of Conconavalin A (Con A) as indicated at the bottom ofthe graph. mCRP1-Fc, mB7RP1, and mB7.2-Fc fusion proteins were added toenriched T-cells from splenocytes in the absence (no adds) or presenceof Con A. 200,000 cells were used in the T-cell proliferation assays ina 96-well plate. Cells were incubated with media (no adds) or Fc fusionproteins as indicated in the graph legend. After 42 hr, cells werepulsed with H-thymidine for 6 hr, then harvested and incorporatedradioactivity determined. Average CPM and standard deviation fromtriplicate samples are represented.

FIG. 10. A) Normal mesenteric lymph node from control Mouse #10 showingthe cortex, paracortex and medulla of the node. Hematoxylin-eosin (H&E)stain, 40× magnification. B) Markedly enlarged mesenteric lymph nodefrom WX11 Mouse #40 with prominent follicular hyperplasia (FH),expansion of paracortex and medullary cord hyperplasia (MH). H&E, 40×.C) Close-up of the medullary cords (MC) and sinuses (MS) from themesenteric lymph node of control Mouse #10. Note the small medullarycords composed of mostly small lymphocytes adjacent to medullary sinuseswith fleshy macrophages. H&E, 400×. D. Close-up of the medullary cords(MC) and sinuses (MS) from the mesenteric lymph node of WX11 Mouse #40.Note the markedly thickened medullary cords composed of large numbers ofplasma cells with occasional Russell body cells (arrow). H&E, 400×. E)Normal spleen from control Mouse #10 showing red pulp and white pulpareas with periarteriolar lymphoid sheaths (PALS), 100×. Inset: close-upof the marginal zone surrounding the white pulp with small lymphocytes,macrophages and occasional plasma cells, 400×. F) Spleen from WX11 Mouse#6 with enlarged white pulp areas, including PALS and follicles (arrow),100×. Inset: close-up of the marginal zone with numerous plasma cellsand occasional Russell bodies, 400×. G) Ileum with Peyer's patch fromcontrol Mouse #25 with the interfollicular zone (arrow) flanked by twosecondary follicles, 40×. H) Ileum with Peyer's patch from WX11 Mouse#32with markedly enlarged follicles with prominent germinal centers andinterfollicular tissue (arrow), 40×.

FIG. 11. A) Normal mesenteric lymph node from control Mouse #5 showingthe cortex, paracortex and medulla of the node. Hematoxylin-eosin (H&E)stain, 40× magnification. B) Markedly enlarged mesenteric lymph nodefrom WX11 Mouse #33 with prominent follicular hyperplasia (top: rows ofsecondary follicles in the outer cortex), expansion of the paracortex(center) and medullary cord hyperplasia (bottom). H&E, 40×. C)Immunohistochemical staining of the mesenteric lymph node from controlMouse #10 with anti-B220 antibody (B cell marker). Note the intensely(brown) staining cortical area and thin medullary cords. Immunostainingperformed using the avidin-biotin complex (ABC) immunoperoxidase method(DAB chromogen, hematoxylin counterstain), 40×. D) Immunohistochemicalstaining of the mesenteric lymph node from WX11 Mouse #33 with anti-B220antibody. Note the intensely staining cortical follicles and medullarycords (although the mature plasma cells in the cords are negative forB220), 40×. E) Immunohistochemical staining of the lymph node fromcontrol Mouse #10 with anti-CD3 antibody (T-cell marker). Note theimmunostaining of the paracortical zone of the node, 40×. F)Immunohistochemical staining of the lymph node from WX11 Mouse #33 withanti-CD3 antibody. Note the enlarged, intensely staining paracorticalareas of the node, 40×.

FIG. 12. A) Structure and sequence of the protein coding region of humanB7RP1 (hB7RP1) SEQ ID NO: 16). Predicted signal sequence of hB7RP1 isunderlined at the amino-terminus. Predicted signal peptide cleavagesites are marked by asterisks. Predicted transmembrane sequence isunderlined toward the carboxy-terminus. B) Amino acid alignment of theputative mature hB7RP1 protein (SEQ ID NO: 17) with the mature murineB7RP1 (mB7RP1) protein (SEQ ID NO: 7).

FIG. 13. A) Structure and sequence of the protein coding region of humanCRP1 (hCRP1) (SEQ ID NO: 21). Predicted signal sequence of hCRP1 isunderlined at the amino-terminus. Predicted signal peptide cleavagesites are marked by asterisks. Predicted transmembrane sequence isunderlined toward the carboxy-terminus. B) Amino acid alignment of thehCRP1 protein (SEQ ID NO: 22) with the murine CRP1 (mB7RP1) protein (SEQID NO: 24).

FIG. 14. CRP1 is on resting memory T-cells. Resting splenocytes from 6-7month old mice were double-stained using B7RP1-Fc labeled by anFITC-conjugated anti-human Fc antibody and a PE-conjugated antibody toeither CD44 (FIG. 14A), CD45RB (FIG. 14B), or CD69 (FIG. 14C).

FIG. 15. T-cell co-stimulation by B7RP1-Fc fusion protein. A) T-cellproliferation induced by different quantities of B7RP1-Fc (closedsquares), B7.2-Fc (closed circles), or OPG-Fc fusion protein control(open squares) in conjunction with anti-CD3 antibody. Fusion proteinswere used at various concentrations to coat 96 well plates pre-coatedwith anti-human Fc FAb₂ (12.5 μg/ml) and anti-CD3 antibody (0.9 μg/ml).B7RP1-Fc and B7.2-Fc co-stimulate T-cells in a dose-dependent fashion upto 0.3 μg/ml, at which the maximal effect is achieved. B) T-cellproliferation induced by B7RP1-Fc (closed squares), B7.2-Fc (closedcircles), non-fused Fc (open squares), or no Fc (open circles) inconjunction an anti-CD3 antibody (0.85 μg/ml) and in the presence ofvarious concentrations of a rabbit anti-B7RP1-Fc polyclonal antibody. Fcfusion proteins were used at a concentration of 0.3 μg/ml and were boundto the plates as above. The anti-B7RP1-Fc antibody was raised topurified B7RP1-Fc by subcutaneous injections of antigen emulsified inadjuvant, and then was affinity purified. The antibodies were incubatedfor 30 min with the Fc fusion proteins before the addition of the cells.The anti-B7RP1-Fc antibody specifically inhibits the T-cellproliferation induced by B7RP1-Fc in a dose-dependent fashion.

FIG. 16. Effect of CRP1-Fc and B7RP1-Fc proteins on the incidence (A)and severity (B) of collagen induced arthritis in mice. Collagen inducedarthritis susceptible B10.RIII mice were immunized at the base of thetail with 10 μg porcine collagen type II in CFA. Mice received 100 μg offusion protein twice per week. Fc fusion proteins and control PBStreatment are indicated in the figure legend.

FIG. 17. Proximal Colon in B7RP1-Fc Transgenic Mice. (A) Normal proximalcolon from control Mouse#53F (female) showing the gut wall with mucosa,submucosa, muscularis and serosa. Hematoxylin-eosin (H&E) stain, 40×magnification. (B) Diffusely thickened proximal colon from B7RP1-Fctransgenic Mouse#111F with prominent glandular hypertrophy, fissuringulceration and transmural inflammation. H&E, 40×. (C) Lower power viewof proximal colon (as in panel B) from B7RP1-Fc transgenic Mouse#111Fwith multifocal fissuring ulceration and transmural inflammation. H&E,20×. (D) Close-up of the fissuring ulcer and hypertrophic colonic glandsfrom B7RP1-Fc transgenic Mouse#111F (shown in panels B and C above).Note the lumen with mucopurulent exudate. H&E, 100×. (E) Close-up ofgranulomatous inflammation in the submucosa of B7RP1-Fc transgenicMouse#112F with a multinucleated giant cell surrounded by macrophages,lymphocytes and fewer neutrophils. H&E, 400×. (F) Close-up ofgranulomatous inflammation in the mucosa of B7RP1-Fc transgenic mouse#112F with epithelioid macrophages mixed with lymphocytes, plasma cellsand fewer neutrophils subjacent to mucosal glands. H&E, 400×.

FIG. 18. Distal Colon in B7RP1-Fc Transgenic Mice. (A) Normal distalcolon from control Mouse#53F (female) showing the layers of the gut wallwith mucosa, submucosa, muscularis and serosa. Hematoxylin-eosin (H&E)stain, 40× magnification. (B) Diffusely thickened distal colon fromB7RP1-Fc transgenic Mouse#111F (female) with prominent glandularhypertrophy and hyperplasia and scattered crypt abscesses. H&E, 40×. (C)Diffusely thickened distal colon from B7RP1-Fc transgenic Mouse#55M(male) with prominent glandular hypertrophy and hyperplasia. H&E, 40×.(D) Diffusely thickened distal colon from B7RP1-Fc transgenic Mouse#112F(female) with hypertrophic colonic glands, focal lymphoid aggregates andmany crypt abscesses. H&E, 40×. (E) Immunohistochemical staining of thedistal colon from B7RP1-Fc transgenic Mouse#112F with anti-CD3 antibody(T-cell marker). Note the immunostaining of the superficial mucosa andcolonic lymphoid patch. H&E, 40×. (F) Close-up of the colonic mucosa ofB7RP1-Fc transgenic Mouse#112F with a crypt abscess (arrow) and lymphoidaggregate composed of B220+ B cells (inset). H&E, 100×.

FIG. 19. Small Intestine in B7RP1-Fc Transgenic Mice. (A) Normalduodenum from control Mouse#53F (female) showing the lumen, villi andcrypts of the mucosa and underlying submucosa, muscularis and serosa.Hematoxylin-eosin (H&E) stain, 40× magnification. (B) Diffuselythickened duodenum from B7RP1-Fc transgenic Mouse#51F (female) withprominent crypt hypertrophy and hyperplasia and mild lymphoplasmacyticinfiltrate in the lamina propria. H&E, 40×. (C) Normal jejunum fromcontrol mouse #53F (female) showing the normal length of the villi andcrypts in the jejunal mucosa. H&E, 40× magnification. (D) Markedlythickened jejunal mucosa from B7RP1-Fc transgenic Mouse#51F (female)with locally extensive crypt hypertrophy and hyperplasia. H&E, 40×. (E)Normal ileum from control Mouse#53F (female) showing the normal lengthof the villi and crypts in the ileal mucosa. H&E, 40× magnification. (F)Mild atrophy of ileal mucosa from B7RP1-Fc transgenic Mouse#231M (male)with focal loss and blunting of villi. H&E, 40×.

FIG. 20. The B7RP1-Fc fusion protein inhibits tumor growth in mice. MethA sarcoma cells were implanted intradermally in the abdomen of Balb/Cmice. On days 7, 10, 14, and 17, after implantation, the mice weretreated with vehicle (dark diamonds) or murine B7RP1-Fc (gray triangles,Example 7). Tumor volume was measured, as described in Example 20, onthe indicated days after implantation. The tumor growth was monitored upto day 28. Each group had eight mice.

FIG. 21. T-cell co-stimulation by human B7RP1-Fc. Anti-CD3 and humanB7RP1 Fc were used to coat 96 well plates, and 1×10⁵ T-cells/well (>98%CD3+) were cultured and harvested as described in Example 21. A)Co-stimulation induced by anti-CD3 only (closed circles), 0.5 μg/mlB7RP1 Fc (closed triangles), 0.5 μg/ml OPG-Fc (open circles), and 5μg/ml anti-CD28 (open triangles) at different concentrations of anti-CD3primary stimulation. Data show that B7RP1-Fc co-stimulated anti-CD3primed T-cells to similar levels as co-stimulation using anti-CD 28antibodies. Data shown are mean [³H]TdR incorporated +/−SD in triplicatewells from one representative experiment of several experimentsgenerated with T-cells isolated from three normal donors. B)Dose-dependent inhibition of B7RP1-Fc co-stimulation by CRP1-Fc. T-cellswere cultured in wells coated with both anti-CD3 at 0.3 μg/ml and 0.5μg/ml B7RP1-Fc. Serially diluted concentrations of CRP1-Fc (closedcircles) or OPG-Fc (open circles) were preincubated with the B7RP1-Fcfor 30 min prior to the addition of T-cells. Data show that CRP1-Fcinhibits B7RP1 induced co-stimulation in a dose-dependent manner.Percent inhibition is plotted against CRP1-Fc or OPG-Fc proteinconcentration. Data shown are mean [³H]TdR incorporated +/−SD of threeexperiments done in triplicate wells and are representative ofexperiments generated with two normal donors. C) Co-stimulation by CHOhuman B7RP1 cells. T-cells were purified from peripheral blood and werecultured with various concentrations of anti-CD3 in the presence ofanti-CD3 alone (closed circles), 1×10⁴ CHO vector control cells (opencircles) or 1×10⁴ CHO B7RP1 cells (closed triangles), as described inExample 22. The data show that membrane-bound B7RP1 co-stimulated T-cellgrowth to a level similar to that observed using B7RP1-Fc fusionproteins. Data shown are the mean+/−SD of triplicate cultures and arerepresentative of results generated with two normal donors. D) Cytokineproduction. T-cells were cultured as described in (FIG. 21A) andsupernatants were collected at 48 (black bars) and 72 (gray bar) hrs.Data show that the amount of IL-2 produced by B7RP1-Fc co-stimulatedcells (top graph) was similar to that produced by cells stimulated byanti-CD3 and control Fc, but significantly less than that produced byanti-CD28 co-stimulated cells. Data also show that B7RP1-Fcco-stimulation enhanced IL-10 (middle graph) and IFN-gamma (bottomgraph) production.

FIG. 22 shows IgE levels in control and B7RP1 transgenic mice.

FIG. 23 shows the combined effects of B7RP1-Fc and B7.2-Fc on chronichypersensitivity. Mice were challenged with oxazolone as described inExample 24. B7RP1-Fc, B7.2-Fc, B7RP1-Fc+B7.2-Fc, and Fc were given athigh (2 or 1+1 mg/Kg, A) and low (0.4 or 0.2+0.2 mg/Kg, B) dose aroundthe time of challenge. A, Compared to Fc, B7RP1-Fc B7.2-Fc, andB7RP1-Fc+B7.2-Fc increased ΔET from day 3 (p<0.001). B7RP1-Fc andB7RP1-Fc+B7.2-Fc increased ΔET more than B7.2-Fc from day 4 (p<0.001).B7RP1-Fc+B7.2-Fc increased ΔET more than B7RP1-Fc from day 4 (p<0.001).B, Compared to Fc, B7RP1-Fc and B7RP1-Fc+B7.2-Fc increased ΔET from day4 (p<0.001). B7RP1-Fc and B7RP1-Fc+B7.2-Fc increased ΔET more thanB7.2-Fc (p<0.001).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for novel polypeptides referred to herein as CRP1and B7RP1, which comprise a receptor-ligand pair that is involved inT-cell activation. cDNAs encoding the polypeptides were identified froma library prepared from mouse intestinal intraepithelial cells andscreened on the basis of homology to the CD28 and CTLA-4 polypeptides(for CRP1) or B7.1 and B7.2 polypeptides (for B7RP1).

CD28 related protein-1, or CRP1, is predicted to be a type Itransmembrane protein with a signal sequence and extracellular domain atthe amino-terminus, a transmembrane domain, and a carboxy terminalintracellular domain (FIG. 1). The full-length CRP1 protein is 180 aminoacids in its mature form. The predicted leader sequence spans aboutamino acid residues 1-20 (relative to the initiating methionine) and theextracellular domain of the mature protein encompasses about residues21-145 (Example 1). The predicted transmembrane domain spans aboutresidues 146-163 and the intracellular domain encompasses about residues164-200. The amino terminal extracellular domain is similar to an Igloop with conserved putative intra- and inter-molecular bondingcysteines. Furthermore, a “MYPPPY” motif, which is previously known tobe important for B7.1 and B7.2 binding to CD28 and CTLA-4, is alsopartially conserved.

CD28 and CTLA-4 are weakly homologous as exemplified by the 26% aminoacid identity between murine CD28 and CTLA-4. There is 19% amino acididentity of CRP1 with murine CD28 and 14% identity of CRP1 with murineCTLA-4. However, critical cysteine residues are conserved between murineCD28, CTLA-4 and CRP1 at residues 42, 63, 83, 109, and 137 (relative tothe initiating methionine in the CRP1 protein, See FIG. 1A). Theapproximate mature protein lengths and locations of the transmembraneregion relative to the carboxy terminus are also similar in CRP1, CD28,and CTLA-4.

Human CRP1 is a transmembrane protein having the nucleotide and aminoacid sequence as shown in FIG. 13A. The predicted leader sequence spansabout residues 1-19 or about residues 1-20. The predicted mature aminoterminus is at residues 20 or 21. Preferably, the mature amino terminusis at position 21. The extracellular domain spans from any of theprediced mature amino termini to about amino acid residue 140, thetransmembrane domain spans about residues 141-161 and the intracellulardomain spans about residues 162-199. Human CRP1 protein has 69% identityto the murine protein and the corresponding nucleotide sequences are 77%identical. The sequence of human CRP1 was reported in Hutloff et al.Nature 397, 263-266 (1999).

B7 related protein-1, or B7RP1, is predicted to be a type Itransmembrane protein with a signal sequence and extracellular domain atthe amino-terminus, a transmembrane domain, and a carboxy terminalintracellular domain (FIG. 2A). The full-length B7RP1 protein is 276amino acids in its mature form. The predicted leader sequence spansabout amino acid residues 1-46 (relative to the initiating methionine)and the extracellular domain of the mature protein encompasses residues47-279 (Example 3). The predicted transmembrane domain spans residues280-298 and the intracellular domain encompasses residues 299-322.Similar to B7.1 and B7.2, the extracellular domain of B7RP1 comprisestwo Ig loops.

B7.1 and B7.2 are weakly homologous as exemplified by the 24% amino acididentity between murine B7.1 and B7.2. There is 20% amino acid identityof B7RP1 with murine B7.1 and 19% identity of B7RP1 with murine B7.2.However, critical cysteine residues are conserved between murine B7.1,B7.2 and B7RP1 at residues 62, 138, 185, and 242 (relative to theinitiating methionine in the B7RP1 protein, FIG. 2A). The approximatemature protein length and location of the transmembrane region relativeto the carboxy terminus are also similar in mB7RP1, B7.1, and B7.2.

Human B7RP1 is also a transmembrane protein with conserved cysteineresidue in the extracellular domain which are necessary for Ig loopstructures. The predicted leader sequence encompasses about residues1-18, 1-19, 1-20, 1-21, 1-23 or 1-27 as shown in FIG. 3A. The predictedmature amino terminus may be at any of the residues 19, 20, 21, 22, 24or 28. Preferably, the amino terminus is at position 19. Theextracellular domain spans from any of the mature amino termini to aboutamino acid residue 259. The predicted transmembrane domain spans aboutresidues 259-274. The intracellular domain encompasses residues 275-302.The full-length human B7RP1 nucleotide and amino acid sequence is shownin FIG. 12A. Human B7RP1 is about 43% identical to the murine protein.

CRP1 and B7RP1 bind each other, but CRP1 does not detectably bind to theB7RP1 related protein B7.2; and B7RP1 does not exhibit detectablebinding to CRP1-related CD28 or CTLA-4 (Example 8). B7RP1 was shown toregulate T-cell proliferation, presumably through the interaction ofB7RP1 with CRP1 receptors (Example 11). Thus, CRP1 and B7RP1 represent anovel pathway for regulating T-cell proliferation and activation.

The interaction of B7RP1 with CRP1 can be regulated in such a mannerthat immune costimulation and T-cell proliferation and activation can beincreased or decreased. By way of example, anti-B7RP1 monoclonal andpolyclonal antibodies raised against murine B7RP1 blocked the B7RP1/CRP1interaction and also blocked T-cell proliferation induced by a B7RP1-Fcfusion protein (see Example 17). A human CRP1-Fc fusion protein blockedhuman T-cell proliferation induced by human B7RP1-Fc (Example 21). Inaddition, addition of a CRP1-Fc fusion protein delayed the onset ofarthritic symptoms in a mouse model of rheumatoid arthritis (see Example18). B7RP1/CRP1 co-stimulation can also be increased by addition ofB7RP1-Fc fusion protein or other activators of this pathway (Example20).

Nucleic Acid Molecules

The term “isolated nucleic acid molecule” refers to a nucleic acidmolecule that is free from at least one contaminating nucleic acidmolecule with which it is naturally associated, and preferablysubstantially free from any other contaminating mammalian nucleic acidmolecules.

The term “allelic variant” refers to one of several possible naturallyoccurring alternate forms of a gene occupying a given locus on achromosome of an organism.

The term “splice variant” refers to a nucleic acid molecule, usuallyRNA, which is generated by alternative processing of intron sequences inan RNA transcript.

The term “high stringency conditions” refers to those conditions which:(1) employ low ionic strength and high temperature for washing, forexample, 0.1×SSC (0.015 M NaCl/0.0015 M sodium citrate) 0.1% NaDodSO₄(SDS) at 50° C., or (2) employ during hybridization a denaturing agentsuch as formamide, for example, 50% (vol/vol) formamide with 0.1% bovineserum albumin/0.1%. Ficoll/0.1% polyvinylpyrrolidone/50 mM sodiumphosphate buffer at pH 6.5 with 5×SSC (750 mM NaCl, 75 mM sodiumcitrate) at 42° C. Another example of high stringency conditions is 50%formamide, 5×SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS.

The term “moderate stringency conditions” refers to those conditionswhich include the use of a washing solution and hybridization conditions(e.g., temperature and ionic strength) less stringent than describedabove. An example of moderately stringent conditions are conditions suchas overnight incubation at 37° C. in a solution comprising: 20%formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μl/ml denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.The skilled artisan will recognize how to adjust the temperature, ionicstrength and other parameters as necessary to accommodate factors suchas probe length and the like.

Recombinant DNA technology methods are set forth in Sambrook et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989)) and/or Ausubel et al., eds.,(Current Protocols in Molecular Biology, Green Publishers Inc. and Wileyand Sons, NY (1994)) which are hereby incorporated by reference in theirentirety.

The invention provides for isolated nucleic acid molecules encoding CRP1and B7RP1 polypeptides. Also provided for are nucleic acid moleculeswhich are fragments, allelic variants, splice variants, or arecomplementary in sequence to molecules encoding CRP1 and B7RP1polypeptides. Nucleic acid molecules which are at least about 70%identical to molecules encoding CRP1 or B7RP1 or which hybridize tomolecules encoding CRP1 or B7RP1 under moderate or high stringencyconditions are also encompassed. The nucleic acid molecules may be cDNA,genomic DNA, RNA or a partially or totally synthetic nucleic acidmolecule. In preferred embodiments, nucleic acid molecules of theinvention are at least about 75%, 80%, 85%, 90% or 95% identical tonucleic acid molecules encoding CRP1 or B7RP1.

A gene or cDNA encoding a CRP1 or B7RP1 polypeptide or fragment thereofmay be obtained, for example, by hybridization screening or PCRamplification of a genomic or cDNA library. Probes or primers useful forscreening the library can be generated based on sequence information forother known genes or gene fragments from the same or a related family ofgenes, for example, conserved motifs found in CRP1 or B7RP1 relatedpolypeptides such as a conserved array of cysteine residues. Inaddition, where a gene encoding a CRP1 or B7RP1 polypeptide has beenidentified from one species, all or a portion of that gene may be usedas a probe to identify homologous genes from other species. The probesor primers may be used to screen cDNA libraries from various tissuesources believed to express the CRP1 or B7RP1 gene.

Where oligonucleotide probes are used to screen cDNA or genomiclibraries, one of the following two high stringency solutions may beused. The first of these is 6×SSC with 0.05% sodium pyrophosphate at 35°C.-62° C., with the temperature depending on the length of theoligonucleotide probe. For example, 14 base pair probes are washed at35-40° C., 17 base pair probes are washed at 45-50° C., 20 base pairprobes are washed at 52-57° C., and 23 base pair probes are washed at57-63° C. The temperature can be increased 2-3° C. where the backgroundnon-specific binding appears high. A second high stringency solutionutilizes tetramethylammonium chloride (TMAC) for washing oligonucleotideprobes. One stringent washing solution is 3 M TMAC, 50 mM Tris-HCl, pH8.0, and 0.2% SDS. The washing temperature using this solution is afunction of the length of the probe. For example, a 17 base pair probeis washed at about 45-50° C.

Another means to prepare a gene encoding a CRP1 or B7RP1 polypeptide orfragment thereof is to employ chemical synthesis using methods wellknown to the skilled artisan such as those described by Engels et al.(Agnew. Chem. Intl. Ed., 28:716-734 (1989)). These methods include,inter alia, the phosphotriester, phosphoramidite, and H-phosphonatemethods for nucleic acid synthesis. A preferred method for such chemicalsynthesis is polymer-supported synthesis using standard phosphoramiditechemistry. Typically, DNA encoding a CRP1 or B7RP1 polypeptide will beseveral hundred nucleotides in length. Nucleic acids larger than about100 nucleotides can be synthesized as several fragments using thesemethods. The fragments can then be ligated together to form a fulllength CRP1 or B7RP1 polypeptide. Usually, the DNA fragment encoding theamino terminus of the polypeptide will have an ATG, which encodes amethionine residue. This methionine may or may not be present on themature form of a CRP1 or B7RP1 polypeptide, depending on whether thepolypeptide produced in the host cell is designed to be secreted fromthat cell.

CRP1 or B7RP1 nucleic acid molecules, fragments, and/or derivatives thatdo not themselves encode polypeptides that are biologically active maynonetheless be useful as hybridization probes in diagnostic assays totest, either qualitatively or quantitatively, for the presence of CRP1or B7RP1 DNA or corresponding RNA in mammalian tissue or bodily fluidsamples.

In some cases, it may be desirable to prepare nucleic acid and/or aminoacid variants of naturally occurring CRP1 or B7RP1 polypeptides. Nucleicacid variants may be produced using site directed mutagenesis, PCRamplification, or other appropriate methods, where the primer(s) havethe desired point mutations (see Sambrook et al., supra, and Ausubel etal., supra, for descriptions of mutagenesis techniques). Chemicalsynthesis using methods described by Engels et al., supra, may also beused to prepare such variants. Other methods known to the skilledartisan may be used as well.

Preferred nucleic acid variants include those containing codons whichhave been altered for optimal expression of CRP1 and B7RP1 polypeptidesin a given host cell. Particular codon alterations will depend upon theselection of protein and host cell. Such “codon optimization” can in oneinstance, be carried out by selecting codons which are preferentiallyused in highly expressed genes in a given host cell. Computer algorithmswhich incorporate codon frequency tables such as “Ecohigh. Cod” forcodon preference of highly expressed bacterial genes may be used and areprovided by the University of Wisconsin Package Version 9.0, GeneticsComputer Group, Madison, Wis. Other useful codon frequency tablesinclude “Celegans_high.cod”, “Celegans_low.cod”, “Drosophila_high.cod”,“Human_high.cod”, “Maize_high.cod”, and “Yeast_high.cod”. Otherpreferred variants are those encoding conservative amino acid changes asdescribed below (e.g., wherein the charge or polarity of the naturallyoccurring amino acid side chain is not altered substantially bysubstitution with a different amino acid) as compared to wild type,and/or those designed to either generate a novel glycosylation and/orphosphorylation site(s), or those designed to delete an existingglycosylation and/or phosphorylation site(s).

The gene, cDNA, or fragment thereof encoding a CRP1 or B7RP1 polypeptidecan be inserted into an appropriate expression or amplification vectorusing standard ligation techniques. The vector is typically selected tobe functional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). The gene, cDNA orfragment thereof encoding the CRP1 or B7RP1 polypeptide may beamplified/expressed in prokaryotic, yeast, insect (baculovirus systems)and/or eukaryotic host cells. Selection of the host cell will depend inpart on whether the CRP1 or B7RP1 polypeptide or fragment thereof is tobe glycosylated and/or phosphorylated. If so, yeast, insect, ormammalian host cells are preferable.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and cloning and expression of insertednucleotide sequences. Such sequences, referred to collectively as“flanking sequences”, will include a promoter and other regulatoryelements such as an enhancer(s), an origin of replication element, atranscriptional termination element, a complete intron sequencecontaining a donor and acceptor splice site, a signal peptide sequence,a ribosome binding site element, a polyadenylation sequence, apolylinker region for inserting the nucleic acid encoding thepolypeptide to be expressed, and a selectable marker element. Each ofthese elements is discussed below. Optionally, the vector may contain a“tag” sequence, i.e., an oligonucleotide molecule located at the 5′ or3′ end of a CRP1 or B7RP1 polypeptide coding sequence; theoligonucleotide molecule encodes polyHis (such as hexaHis), or other“tag” such as FLAG, HA (hemaglutinin Influenza virus) or myc for whichcommercially available antibodies exist. This tag is typically fused tothe polypeptide upon expression of the polypeptide, and can serve asmeans for affinity purification of a CRP1 or B7RP1 polypeptide from thehost cell. Affinity purification can be accomplished, for example, bycolumn chromatography using antibodies against the tag as an affinitymatrix. Optionally, the tag can subsequently be removed from a purifiedCRP1 or B7RP1 polypeptide by various means such as using certainpeptidases.

The human immunoglobulin hinge and Fc region may be fused at either theN-terminus or C-terminus of a CRP1 or B7RP1 polypeptide by one skilledin the art. The subsequent Fc-fusion protein can be purified by use of aProtein A affinity column. An immunoglobin Fc region is known to exhibita long pharmacokinetic half-life in vivo and proteins fused to an Fcregion have been found to exhibit a substantially greater half-life invivo compared to the unfused counterpart.

Also, fusion to the Fc region allows for dimerization and/ormultimerization of the molecule that may be useful for the bioactivityof some molecules. The flanking sequence may be homologous (i.e., fromthe same species and/or strain as the host cell), heterologous (i.e.,from a species other than the host cell species or strain), hybrid(i.e., a combination of flanking sequences from more than one source),synthetic, or it may be the native CRP1 or B7RP1 nucleic acid flankingsequences. As such, the source of the flanking sequence may be anyunicellular prokaryotic or eukaryotic organism, any vertebrate orinvertebrate organism, or any plant, provided that the flanking sequenceis functional in, and can be activated by, the host cell machinery.

The flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein other than CRP1 or B7RP1 nucleic acidflanking sequence will have been previously identified by mapping and/orby restriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of the flanking sequence may beknown. Here, the flanking sequence may be synthesized using the methodsdescribed above for nucleic acid synthesis or cloning.

Where all or only a portion of the flanking sequence is known, it may beobtained using PCR and/or by screening a genomic library with suitableoligonucleotide and/or flanking sequence fragments from the same oranother species.

Where the flanking sequence is not known, a fragment of DNA containing aflanking sequence may be isolated from a larger piece of DNA that maycontain, for example, a coding sequence or even another gene or genes.Isolation may be accomplished by restriction endonuclease digestionusing one or more carefully selected enzymes to isolate the proper DNAfragment. After digestion, the desired fragment may be isolated byagarose gel purification, Qiagen® column or other methods known to theskilled artisan. Selection of suitable enzymes to accomplish thispurpose will be readily apparent to one of ordinary skill in the art.

The origin of replication element is typically a part of prokaryoticexpression vectors purchased commercially, and aids in the amplificationof the vector in a host cell. Amplification of the vector to a certaincopy number can, in some cases, be important for optimal expression ofthe CRP1 or B7RP1 polypeptide. If the vector of choice does not containan origin of replication site, one may be chemically synthesized basedon a known sequence, and ligated into the vector.

The transcription termination element is typically located 3′ of the endof a CRP1 or B7RP1 polypeptide coding sequence and serves to terminatetranscription of a CRP1 or B7RP1 polypeptide. Usually, the transcriptiontermination element in prokaryotic cells is a G-C rich fragment followedby a poly T sequence. While the element is easily cloned from a libraryor even purchased commercially as part of a vector, it can also bereadily synthesized using methods for nucleic acid synthesis such asthose described above.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarnosequence (prokaryotes) or the Kozak sequence (eukaryotes), is usuallynecessary for translational initiation of mRNA. The element is typicallylocated 3′ to the promoter and 5′ to the coding sequence of the CRP1 orB7RP1 polypeptide to be synthesized. The Shine-Dalgarno sequence isvaried but is typically a polypurine (i.e., having a high A-G content).Many Shine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector.

In those cases where it is desirable for a CRP1 or B7RP1 polypeptide tobe secreted from the host cell, a signal sequence may be used to directexport of the polypeptide from the host cell. A CRP1 or B7RP1transmembrane domain is also inactivated by mutation or deletion toprevent attachment to the host membrane. Typically, the signal sequenceis positioned in the coding region of a CRP1 or B7RP1 gene or cDNA, ordirectly at the 5′ end of a CRP1 or B7RP1 gene coding region. Manysignal sequences have been identified, and any of them that arefunctional in the selected host cell may be used in conjunction with aCRP1 or B7RP1 gene or cDNA. Therefore, the signal sequence may behomologous or heterologous to a CRP1 or B7RP1 gene or cDNA, and may behomologous or heterologous to a CRP1 or B7RP1 polypeptides gene or cDNA.Additionally, the signal sequence may be chemically synthesized usingmethods set forth above.

In most cases, secretion of the polypeptide from the host cell via thepresence of a signal peptide will result in the removal of the aminoterminal methionine from the polypeptide.

In many cases, transcription of a CRP1 or B7RP1 gene or cDNA isincreased by the presence of one or more introns in the vector; this isparticularly true where a CRP1 or B7RP1 polypeptide is produced ineukaryotic host cells, especially mammalian host cells. The introns usedmay be naturally occurring within a CRP1 or B7RP1 gene, especially wherethe gene used is a full length genomic sequence or a fragment thereof.Where the intron is not naturally occurring within the gene (as for mostcDNAs), the intron(s) may be obtained from another source. The positionof the intron with respect to the 5′ flanking sequence and a CRP1 orB7RP1 gene is generally important, as the intron must be transcribed tobe effective. As such, where a CRP1 or B7RP1 gene inserted into theexpression vector is a cDNA molecule, the preferred position for theintron is 3′ to the transcription start site, and 5′ to the polyAtranscription termination sequence. Preferably the intron or intronswill be located on one side or the other (i.e., 5′ or 3′) of the cDNAsuch that it does not interrupt the this coding sequence. Any intronfrom any source, including any viral, prokaryotic and eukaryotic (plantor animal) organisms, may be used to practice this invention, providedthat it is compatible with the host cell(s) into which it is inserted.Also included herein are synthetic introns. Optionally, more than oneintron may be used in the vector.

Where one or more of the elements set forth above are not alreadypresent in the vector to be used, they may be individually obtained andligated into the vector. Methods used for obtaining each of the elementsare well known to the skilled artisan.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (InvitrogenCompany, San Diego, Calif.), pBSII (Stratagene Company, La Jolla,Calif.), pET15b (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL(BlueBacII; Invitrogen), and pFastBacDual (Gibco/BRL, Grand Island,N.Y.).

After the vector has been constructed and a nucleic acid moleculeencoding a full length or truncated CRP1 or B7RP1 polypeptide has beeninserted into the proper site of the vector, the completed vector may beinserted into a suitable host cell for amplification and/or polypeptideexpression.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as a yeast cell, an insect cell, or a vertebrate cell).The host cell, when cultured under appropriate conditions, cansynthesize a CRP1 or B7RP1 polypeptide which can subsequently becollected from the culture medium (if the host cell secretes it into themedium) or directly from the host cell producing it (if it is notsecreted). After collection, a CRP1 or B7RP1 polypeptide can be purifiedusing methods such as molecular sieve chromatography, affinitychromatography, and the like.

Selection of the appropriate host cell for CRP1 or B7RP1 polypeptideproduction will depend on various factors, such as desired expressionlevels, polypeptide modifications that are required for activity, suchas glycosylation or phosphorylation, or ease of folding into abiologically active molecule.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO), human embryonic kidney (HEK) 293 or293T-cells, or 3T3 cells. The selection of suitable mammalian host cellsand methods for transformation, culture, amplification, screening andproduct production and purification are known in the art. Other suitablemammalian cell lines, are the monkey COS-1 and COS-7 cell lines, and theCV-1 cell line. Further exemplary mammalian host cells include primatecell lines and rodent cell lines, including transformed cell lines.Normal diploid cells, cell strains derived from in vitro culture ofprimary tissue, as well as primary explants, are also suitable.Candidate cells may be genotypically deficient in the selection gene, ormay contain a dominantly acting selection gene. Other suitable mammaliancell lines include but are not limited to, mouse neuroblastoma N2Acells, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c orNIH mice, BHK or HaK hamster cell lines.

Similarly useful as host cells are bacterial cells. For example, thevarious strains of E. coli (e.g., HB101, DH5α, DH10, and MC1061) arewell-known as host cells in the field of biotechnology. Various strainsof B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomycesspp., and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described for examplein Kitts et al. (Biotechniques, 14:810-817 (1993)), Lucklow (Curr. Opin.Biotechnol., 4:564-572 (1993)) and Lucklow et al. (J. Virol.,67:4566-4579 (1993)). Preferred insect cells are Sf-9 and Hi5(Invitrogen, Carlsbad, Calif.).

“Transformation” or “transfection” of an expression vector into theselected host cell may be accomplished using such methods as calciumchloride, electroporation, microinjection, lipofection or theDEAE-dextran method. The method selected will in part be a function ofthe type of host cell to be used. These methods and other suitablemethods are well known to the skilled artisan, and are set forth, forexample, in Sambrook et al., supra.

The host cells transformed or transfected with an expression vector maybe cultured using standard media well known to the skilled artisan. Themedia will usually contain all nutrients necessary for the growth andsurvival of the cells. Suitable media for culturing E. coli cells are,for example, Luria Broth (LB) and/or Terrific Broth (TB). Suitable mediafor culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all of whichmay be supplemented with serum and/or growth factors as required by theparticular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of CRP1 or B7RP1 polypeptide produced in the host cell can beevaluated using standard methods known in the art. Such methods include,without limitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or activity assays such as DNA binding gelshift assays.

If a CRP1 or B7RP1 polypeptide has been designed to be secreted from thehost cells, the majority of polypeptide may be found in the cell culturemedium. Polypeptides prepared in this way will typically not possess anamino terminal methionine, as it is removed during secretion from thecell. If, however, a CRP1 or B7RP1 polypeptide is not secreted from thehost cells, it will be present in the cytoplasm and/or the nucleus (foreukaryotic host cells) or in the cytosol (for gram negative bacteriahost cells) and may have an amino terminal methionine.

Purification of a CRP1 or B7RP1 polypeptide from solution can beaccomplished using a variety of techniques. If the polypeptide has beensynthesized such that it contains a tag such as Hexahistidine (CRP1 orB7RP1/hexaHis) or other small peptide such as FLAG (Eastman Kodak Co.,New Haven, Conn.) or myc (Invitrogen, Carlsbad, Calif.) at either itscarboxyl or amino terminus, it may essentially be purified in a one-stepprocess by passing the tagged polypeptide through an affinity columnwhere the column matrix has a high affinity for the tag or for thepolypeptide directly (i.e., a monoclonal antibody specificallyrecognizing a CRP1 or B7RP1 polypeptide). For example, polyhistidinebinds with great affinity and specificity to nickel, thus an affinitycolumn of nickel (such as the Qiagen® nickel columns) can be used forpurification of CRP1 or B7RP1/polyHis. (See for example, Ausubel et al.,eds., Current Protocols in Molecular Biology, Section 10.11.8, JohnWiley & Sons, New York (1993)).

Where a CRP1 or B7RP1 polypeptide is prepared without a tag attached,and no antibodies are available, other well known procedures forpurification can be used. Such procedures include, without limitation,ion exchange chromatography, molecular sieve chromatography, HPLC,native gel electrophoresis in combination with gel elution, andpreparative isoelectric focusing (“Isoprime” machine/technique, HoeferScientific). In some cases, two or more of these techniques may becombined to achieve increased purity.

If it is anticipated that a CRP1 or B7RP1 polypeptide will be foundprimarily intracellularly, the intracellular material (includinginclusion bodies for gram-negative bacteria) can be extracted from thehost cell using any standard technique known to the skilled artisan. Forexample, the host cells can be lysed to release the contents of theperiplasm/cytoplasm by French press, homogenization, and/or sonicationfollowed by centrifugation.

If a CRP1 or B7RP1 polypeptide has formed inclusion bodies in thecytosol, the inclusion bodies can often bind to the inner and/or outercellular membranes and thus will be found primarily in the pelletmaterial after centrifugation. The pellet material can then be treatedat pH extremes or with chaotropic agent such as a detergent, guanidine,guanidine derivatives, urea, or urea derivatives in the presence of areducing agent such as dithiothreitol at alkaline pH or triscarboxyethyl phosphine at acid pH to release, break apart, andsolubilize the inclusion bodies. The polypeptide in its now soluble formcan then be analyzed using gel electrophoresis, immunoprecipitation orthe like. If it is desired to isolate a CRP1 or B7RP1 polypeptide,isolation may be accomplished using standard methods such as those setforth below and in Marston et al. (Meth. Enz., 182:264-275 (1990)). Insome cases, a CRP1 or B7RP1 polypeptide may not be biologically activeupon isolation. Various methods for “refolding” or converting thepolypeptide to its tertiary structure and generating disulfide linkagescan be used to restore biological activity. Such methods includecontacting the solubilized polypeptide with a solution having a pHusually above 7 and in the presence of a particular concentration of anappropriate chaotrope. In most cases the refolding/oxidation solutionwill also contain a reducing agent or the reducing agent and thecorresponding oxidized form in a specific ratio to generate a particularredox potential allowing for disulfide shuffling to occur in theformation of the protein's cysteine bridge(s). Some of the commonly usedredox couples include cysteine/cystamine, glutathione (GSH)/dithiobisGSH, cupric chloride, dithiothreitol (DTT)/dithiane DTT,2-mercaptoethanol (bME)/dithio-b (ME). In many instances a cosolvent isnecessary to increase the efficiency of the refolding and the morecommon reagents used for this purpose include glycerol, polyethyleneglycol of various molecular weights, and arginine.

CRP1 or B7RP1 polypeptides, fragments, and/or derivatives thereof mayalso be prepared by chemical synthesis methods (such as solid phasepeptide synthesis) using techniques known in the art such as those setforth by Merrifield et al., (J. Am. Chem. Soc., 85:2149 (1963)),Houghten et al. (Proc Natl Acad. Sci. USA, 82:5132 (1985)), and Stewartand Young (Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford,Ill. (1984)). Such polypeptides may be synthesized with or without amethionine on the amino terminus. Chemically synthesized CRP1 or B7RP1polypeptides or fragments may be oxidized using methods set forth inthese references to form disulfide bridges. CRP1 or B7RP1 polypeptidesor fragments are expected to have biological activity comparable to CRP1or B7RP1 polypeptides produced recombinantly or purified from naturalsources, and thus may be used interchangeably with recombinant ornatural CRP1 or B7RP1 polypeptide.

Polypeptides

The term “CRP1 or B7RP1 polypeptide” refers to a polypeptide having theamino acid sequence of FIG. 1A (SEQ ID NO:2), FIG. 2A (SEQ ID NO:7) orFIG. 3A (SEQ ID NO:12) and all related polypeptides described herein.Related polypeptides includes allelic variants, splice variants,fragments, derivatives, substitution, deletion, and insertion variants,fusion polypeptides, and orthologs. Such related polypeptides may bemature polypeptides, i.e., polypeptide lacking a signal peptide. A CRP1or B7RP1 polypeptide may or may not have amino terminal methionine,depending on the manner in which they are prepared.

The term “CRP1 or B7RP1 polypeptide fragment” refers to a peptide orpolypeptide that is less than the full length amino acid sequence of aCRP1 or B7RP1 polypeptide as set forth in FIG. 1A (SEQ ID NO:2), FIG. 2A(SEQ ID NO:7) or FIG. 3A (SEQ ID NO:12). Such a fragment may result fromtruncation at the amino terminus, truncation at the carboxy terminus,and/or a deletion internal to the polypeptide sequence. Such CRP1 orB7RP1 polypeptides fragments may be prepared with or without an aminoterminal methionine. In addition, CRP1 or B7RP1 polypeptides fragmentsmay be naturally-occurring splice variants, other splice variants, andfragments resulting from naturally occurring in vivo protease activity.Preferred CRP1 or B7RP1 polypeptide fragments include soluble forms ofCRP1 or B7RP1 which lack a functional transmembrane domain and comprisepart or all of the extracellular domain of either CRP1 or B7RP1.

The term “CRP1 or B7RP1 polypeptide variants” refers to CRP1 or B7RP1polypeptides whose amino acid sequences contain one or more amino acidsequence substitutions, deletions, and/or additions as compared to theCRP1 or B7RP1 polypeptides amino acid sequences set forth in FIG. 1A(SEQ ID NO:2), FIG. 2A (SEQ ID NO:7) or FIG. 3A (SEQ ID NO:12). SuchCRP1 or B7RP1 polypeptides variants can be prepared from thecorresponding CRP1 and B7RP1 polypeptides nucleic acid moleculevariants, which have a DNA sequence that varies accordingly from the DNAsequences for CRP1 or B7RP1 polypeptides.

As used herein, the term “CRP1 or B7RP1 polypeptide derivatives” refersto CRP1 or B7RP1 polypeptides, variants, or fragments thereof, that havebeen chemically modified, as for example, by addition of one or morewater soluble polymers, N-linked or O-linked carbohydrates, sugars,phosphates, and/or other such molecules, where the molecule or moleculesare not naturally attached to wild-type CRP1 or B7RP1 polypeptides.Derivatives further includes deletion of one or more chemical groupsnaturally attached to the CRP1 or B7RP1 polypeptide.

As used herein, the terms “biologically active CRP1 or B7RP1polypeptides”, “biologically active CRP1 or B7RP1 polypeptidefragments”, “biologically active CRP1 or B7RP1 polypeptide variants”,and “biologically active CRP1 or B7RP1 polypeptide derivatives” refer toCRP1 or B7RP1 polypeptides having at least one of the activitiescharacteristic of CRP1 or B7RP1. One activity is binding of B7RP1 toCRP1. Another activity is the ability of CRP1 or B7RP1 to stimulateT-cell proliferation and/or activation.

The term “ortholog” refers to a polypeptide that corresponds to apolypeptide identified from a species. For example, mouse and humanB7RP1 polypeptides are considered orthologs.

The term “mature amino acid sequence” refers to a polypeptide lacking aleader sequence.

The term “isolated polypeptide” refers to a polypeptide that is freefrom at least one contaminating polypeptide that is found in its naturalenvironment, and preferably substantially free from any othercontaminating mammalian polypeptides.

The term “identity,” as known in the art, is a relationship between thesequences of two or more nucleic acid molecules or two or morepolypeptides, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or nucleic acid molecule sequences, as the case may be, asdetermined by the match between strings of nucleotide or amino acidsequences. “Identity” measures the percent of identical matches betweentwo or more sequences with gap alignments addressed by particularcomputer programs (i.e., “algorithms”).

The term “similarity” refers to a related concept, but in contrast to“identity”, a measure of similarity includes both identical matches andconservative substitution matches. Since conservative substitutionsapply to polypeptides and not nucleic acid molecules, similarity onlydeals with polypeptide sequence comparisons. If two polypeptidesequences have, for example, 10/20 identical amino acids, and theremainder are all non-conservative substitutions, then the percentidentity and similarity would both be 50%. If in the same example, thereare 5 more positions where there are conservative substitutions, thenthe percent identity remains 50%, but the percent similarity would be75% (15/20). Therefore, in cases where there are conservativesubstitutions, the degree of similarity between two polypeptidesequences will be higher than the percent identity between those twosequences. “Conservative” amino acid substitutions are described hereinbelow in reference to Table I. Based on Table I, conservative amino acidsubstitutions are alternate amino acids selected from the same grouping,e.g., basic, acidic, uncharged polar, and non-polar. For example,conservative amino acid substitutions for arginine would be lysine andhistidine.

Identity and similarity can be readily calculated by known methods,including but not limited to those described in Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M. Stockton Press, New York, 1991; and Carillo, H., andLipman, D., SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine identity and/or similarity are designedto give the largest match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux, J., etal., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group,University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA(Atschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990). The BLAST Xprogram is publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul, S., et al.NCB NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol.215:403-410 (1990). The well known Smith Waterman algorithm may also beused to determine identity.

By way of example, using the computer algorithm GAP (Genetics ComputerGroup, University of Wisconsin, Madison, Wis.), two polypeptides forwhich the percent sequence identity is to be determined are aligned foroptimal matching of their respective amino acids (the “matched span”, asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3× the average diagonal; the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 1/10 times the gap opening penalty), as well as a comparisonmatrix such as PAM 250 or BLOSUM 62 are used in conjunction with thealgorithm. A standard comparison matrix (see Dayhoff et al., in: Atlasof Protein Sequence and Structure, vol. 5, supp. 3 (1978) for the PAM250comparison matrix; see Henikoff et al., Proc. Natl. Acad. Sci. USA,89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also usedby the algorithm.

Preferred parameters for polypeptide sequence comparison include thefollowing:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)

Comparison matrix: BLOSUM 62 from Henikoff and Henikoff, Proc. Natl.Acad. Sci. USA 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

The GAP program is useful with the above parameters. The aforementionedparameters are the default parameters for polypeptide comparisons (alongwith no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparisoninclude the following:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

The GAP program is also useful with the above parameters. Theaforementioned parameters are the default parameters for nucleic acidmolecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused by those of skill in the art, including those set forth in theProgram Manual, Wisconsin Package, Version 9, September, 1997. Theparticular choices to be made will depend on the specific comparison tobe made, such as DNA to DNA, protein to protein, protein to DNA; andadditionally, whether the comparison is between pairs of sequences (inwhich case GAP or BestFit are generally preferred) or between onesequence and a large database of sequences (in which case FASTA orBLASTA are preferred).

Polypeptides that are at least about 70 percent identical will typicallyhave one or more amino acid substitutions, deletions, and/or additionsas compared to a wild type CRP1 or B7RP1 polypeptide. In preferredembodiment, polypeptides will have about 75%, 80%, 85%, 90% or 95%identity to CRP1 or B7RP1 polypeptides. Usually, the substitutions ofthe native residue will be either alanine, or a conservative amino acidso as to have little or no effect on the overall net charge, polarity,or hydrophobicity of the polypeptide. Conservative substitutions are setforth in Table I below.

TABLE I Conservative Amino Acid Substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Uncharged Polar: glutamineasparagine serine threonine tyrosine Non-Polar: phenylalanine tryptophancysteine glycine alanine valine proline methionine leucine norleucineisoleucine

CRP1 or B7RP1 polypeptide derivatives are provided by the invention. Inone embodiment, chemically modified CRP1 or B7RP1 polypeptidecompositions in which CRP1 or B7RP1 polypeptides are linked to a polymerare included within the scope of the present invention. The polymerselected is typically water soluble so that the protein to which it isattached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer selected is usually modified tohave a single reactive group, such as an active ester for acylation oran aldehyde for alkylation, so that the degree of polymerization may becontrolled as provided for in the present methods. The polymer may be ofany molecular weight, and may be branched or unbranched. Included withinthe scope of the invention is a mixture of polymers. Preferably, fortherapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable.

The water soluble polymer or mixture thereof may be selected from thegroup consisting of, for example, polyethylene glycol (PEG),monomethoxy-polyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol.

For the acylation reactions, the polymer(s) selected should have asingle reactive ester group. For reductive alkylation, the polymer(s)selected should have a single reactive aldehyde group. A preferredreactive aldehyde is polyethylene glycol propionaldehyde, which is waterstable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see U.S.Pat. No. 5,252,714).

Pegylation of CRP1 or B7RP1 polypeptides may be carried out by any ofthe pegylation reactions known in the art, as described for example inthe following references: Focus on Growth Factors 3: 4-10 (1992); EP 0154 316; and EP 0 401 384. Preferably, the pegylation is carried out viaan acylation reaction or an alkylation reaction with a reactivepolyethylene glycol molecule (or an analogous reactive water-solublepolymer) as described below.

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol, abbreviated PEG. As used herein, polyethyleneglycol is meant to encompass any of the forms of PEG that have been usedto derivatize other proteins, such as mono-(C1-C10) alkoxy- oraryloxy-polyethylene glycol.

In general, chemical derivatization may be performed under any suitableconditions used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylated CRP1 andB7RP1 polypeptides will generally comprise the steps of (a) reacting thepolypeptide with polyethylene glycol (such as a reactive ester oraldehyde derivative of PEG) under conditions whereby CRP1 or B7RP1polypeptide becomes attached to one or more PEG groups, and (b)obtaining the reaction product(s). In general, the optimal reactionconditions for the acylation reactions will be determined based on knownparameters and the desired result. For example, the larger the ratio ofPEG:protein, the greater the percentage of poly-pegylated product.

Generally, conditions which may be alleviated or modulated byadministration of CRP1 or B7RP1 polymer conjugates include thosedescribed herein for non-conjugated CRP1 or B7RP1 polypeptides. However,the conjugated disclosed herein may have additional activities, enhancedor reduced biological activity, or other characteristics, such asincreased or decreased half-life, as compared to the non-derivatizedmolecules.

CRP1 or B7RP1 polypeptides, fragments variants, and derivatives, may beemployed alone, together, or in combination with other pharmaceuticalcompositions. CRP1 or B7RP1 polypeptides, fragments, variants, andderivatives may be used in combination with cytokines, growth factors,antibiotics, anti-inflammatories, and/or chemotherapeutic agents as isappropriate for the indication being treated.

The invention provides for selective binding agents of CRP1 or B7RP1. Aselective binding agent refers to a molecule having specificity for CRP1or B7RP1 and may include a protein, peptide, nucleic acid, carbohydrate,lipid or small molecular weight compound. A selective binding agentinteracts either with CRP1 or B7RP1 and in turn regulates the binding ofCRP1 to B7RP1. In one embodiment, a selective binding agent partially orcompletely blocks the binding of CRP1 to B7RP1 and partially orcompletely inhibits at least one biological activity of CRP1 or B7RP1,such as immune costimulatory activity. In another embodiment, theselective binding agent is an antibody. The antibody may beimmunoreactive with either CRP1 or B7RP1 and is preferablyimmunoreactive with B7RP1. In yet another embodiment of the invention,an antibody reactive with B7RP1 binds to an eptiope on B7RP1 such thatbinding to CRP1 is partially or completely blocked and at least onebiological activity of B7RP1, such as immune costimulatory activity, ispartially or completely inhibited. The term partially inhibited meansthat at least a detectable level of inhibition has occurred. The termcompletely inhibited means that no further increase in inhibition hasoccurred.

CRP1 or B7RP1 polypeptides, fragments, variants, and/or derivatives maybe used to prepare antibodies using methods known in the art. Thus,antibodies that react with the CRP1 or B7RP1 polypeptides, as well asreactive fragments of such antibodies, are also contemplated as withinthe scope of the present invention. The antibodies may be polyclonal,monoclonal, recombinant, chimeric, single-chain and/or bispecific.Typically, the antibody or fragment thereof will either be of humanorigin, or will be “humanized”, i.e., prepared so as to prevent orminimize an immune reaction to the antibody when administered to apatient. The antibody fragment may be any fragment that is reactive withCRP1 and B7RP1 polypeptides of the present invention, such as, F_(ab),F_(ab′), etc. Also provided by this invention are the hybridomasgenerated by presenting any CRP1 or B7RP1 polypeptide or fragmentsthereof as an antigen to a selected mammal, followed by fusing cells(e.g., spleen cells) of the mammal with certain cancer cells to createimmortalized cell lines by known techniques. The methods employed togenerate such cell lines and antibodies directed against all or portionsof a human CRP1 or B7RP1 polypeptide of the present invention are alsoencompassed by this invention.

Monoclonal antibodies of the invention include “chimeric” antibodies inwhich a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequence in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chains(s) is identical with orhomologous to corresponding sequence in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (U.S. Pat. No. 4,816,567; Morrison, et al., Proc.Natl. Acad. Sci. 81, 6851-6855 (1985)).

In a preferred embodiment, the chimeric anti-CRP 1 or B7RP1 antibody isa “humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.Humanization can be performed following methods known in the art (Jones,et al., Nature 321, 522-525 (1986); Riechmann, et al., Nature, 332,323-327 (1988); Verhoeyen, et al., Science 239, 1534-1536 (1988)), bysubstituting rodent complementarily-determining regions (CDRs) for thecorresponding regions of a human antibody.

Also encompassed by the invention are fully human anti-CRP1 oranti-B7RP1 antibodies. Such antibodies may be produced by immunizationwith a CRP1 or B7RP1 antigen of transgenic animals (e.g., mice) that arecapable of producing a repertoire of human antibodies in the absence ofendogenous immunoglobulin production. See, for example, Jakobovits, etal., Proc. Natl. Acad. Sci. 90, 2551-2555 (1993); Jakobovits, et al.,Nature 362, 255-258 (1993). Human antibodies can also be produced inphage-display libraries (Hoogenboom, et al., J. Mol. Biol. 227, 381(1991); Marks, et al., J. Mol. Biol. 222, 581 (1991).

Selective binding agents of the invention may be used to regulate thebinding of CRP1 to B7RP1 and regulate at least one biological activitymediated by CRP1 and B7RP1 such as immune co-stimulation. An example ofsuch selective binding agents are antibodies immunoreactive with eitherCRP1 or B7RP1. The antibodies may be used therapeutically, such as toinhibit binding of the CRP1 and B7RP1 polypeptide to its bindingpartner. The antibodies may further be used for in vivo and in vitrodiagnostic purposes, such as in labeled form to detect the presence ofCRP1 and B7RP1 polypeptide in a body fluid or cell sample.

Pharmaceutical Compositions and Administration

Pharmaceutical compositions of CRP1 or B7RP1 polypeptides are within thescope of the present invention. Such compositions may comprise atherapeutically effective amount of the polypeptide or fragments,variants, or derivatives in admixture with a pharmaceutically acceptablecarrier. In preferred embodiments, pharmaceutical compositions compriseCRP1 or B7RP1 polypeptides as soluble forms which comprise part or allof a CRP1 or B7RP1 extracellular domain. Typically, a CRP1 and B7RP1polypeptide therapeutic compound will be administered in the form of acomposition comprising purified polypeptide, fragment, variant, orderivative in conjunction with one or more physiologically acceptablecarriers, excipients, or diluents. The carrier material may be water forinjection, preferably supplemented with other materials common insolutions for administration to mammals. Neutral buffered saline orsaline mixed with serum albumin are exemplary appropriate carriers.Preferably, the product is formulated as a lyophilizate usingappropriate excipients (e.g., sucrose). Other standard carriers,diluents, and excipients may be included as desired. Other exemplarycompositions comprise Tris buffer of about pH 7.0-8.5, or acetate bufferof about pH 4.0-5.5, which may further include sorbitol or a suitablesubstitute therefor.

CRP1 or B7RP1 pharmaceutical compositions can be administeredparenterally. Alternatively, the compositions may be administeredintravenously or subcutaneously. When systemically administered, thetherapeutic compositions for use in this invention may be in the form ofa pyrogen-free, parenterally acceptable aqueous solution. Thepreparation of such pharmaceutically acceptable protein solutions, withdue regard to pH, isotonicity, stability and the like, is within theskill of the art.

Therapeutic formulations of CRP1 and B7RP1 polypeptide compositionsuseful for practicing the present invention may be prepared for storageby mixing the selected composition having the desired degree of puritywith optional physiologically acceptable carriers, excipients, orstabilizers (Remington's Pharmaceutical Sciences, 18th Edition, A. R.Gennaro, ed., Mack Publishing Company (1990)) in the form of alyophilized cake or an aqueous solution. Acceptable carriers, excipientsor stabilizers are nontoxic to recipients and are preferably inert atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, or other organic acids; antioxidants such asascorbic acid; low molecular weight polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, pluronics or polyethylene glycol (PEG).

An effective amount of a CRP1 or B7RP1 polypeptide composition(s) to beemployed therapeutically will depend, for example, upon the therapeuticobjectives such as the indication for which the CRP1 and B7RP1polypeptide is being used, the route of administration, and thecondition of the patient. Accordingly, it will be necessary for thetherapist to titer the dosage and modify the route of administration asrequired to obtain the optimal therapeutic effect. A typical dailydosage may range from about 0.1 μg/kg to up to 100 mg/kg or more,depending on the factors mentioned above. Typically, a clinician willadminister the composition until a dosage is reached that achieves thedesired effect. The composition may therefore be administered as asingle dose, or as two or more doses (which may or may not contain thesame amount of a CRP1 or B7RP1 polypeptide) over time, or as acontinuous infusion via implantation device or catheter.

As further studies are conducted, information will emerge regardingappropriate dosage levels for treatment of various conditions in variouspatients, and the ordinary skilled worker, considering the therapeuticcontext, the type of disorder under treatment, the age and generalhealth of the recipient, will be able to ascertain proper dosing.

The CRP1 or B7RP1 polypeptide composition to be used for in vivoadministration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes. Where the compositionis lyophilized, sterilization using these methods may be conductedeither prior to, or following, lyophilization and reconstitution. Thecomposition for parenteral administration ordinarily will be stored inlyophilized form or in solution.

Therapeutic compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

The route of administration of the composition is in accord with knownmethods, e.g. oral, injection or infusion by intravenous,intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraocular, intraarterial, orintralesional routes, or by sustained release systems or implantationdevice which may optionally involve the use of a catheter. Wheredesired, the compositions may be administered continuously by infusion,bolus injection or by implantation device.

Alternatively or additionally, the composition may be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which CRP1 and B7RP1 polypeptide hasbeen absorbed.

Where an implantation device is used, the device may be implanted intoany suitable tissue or organ, and delivery of a CRP1 or B7RP1polypeptide may be directly through the device via bolus, or viacontinuous administration, or via catheter using continuous infusion. ACRP1 or B7RP1 polypeptide may be administered in a sustained releaseformulation or preparation. Suitable examples of sustained-releasepreparations include semipermeable polymer matrices in the form ofshaped articles, e.g. films, or microcapsules. Sustained releasematrices include polyesters, hydrogels, polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al, Biopolymers, 22: 547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15: 167-277 (1981)] and Langer, Chem. Tech., 12: 98-105 (1982)),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also may include liposomes, which can be prepared by any ofseveral methods known in the art (e.g., Eppstein et al., Proc. Natl.Acad. Sci. USA, 82: 3688-3692 (1985); EP 36,676; EP 88,046; EP 143,949).

In some cases, it may be desirable to use CRP1 or B7RP1 polypeptidecompositions in an ex vivo manner. Here, cells, tissues, or organs thathave been removed from the patient are exposed to a CRP1 or B7RP1polypeptide compositions after which the cells, tissues and/or organsare subsequently implanted back into the patient.

In other cases, a CRP1 or B7RP1 polypeptide may be delivered throughimplanting into patients certain cells that have been geneticallyengineered, using methods such as those described herein, to express andsecrete the polypeptides, fragments, variants, or derivatives. Suchcells may be animal or human cells, and may be derived from thepatient's own tissue or from another source, either human or non-human.Optionally, the cells may be immortalized. However, in order to decreasethe chance of an immunological response, it is preferred that the cellsbe encapsulated to avoid infiltration of surrounding tissues. Theencapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow release of the proteinproduct(s) but prevent destruction of the cells by the patient's immunesystem or by other detrimental factors from the surrounding tissues.

Methods used for membrane encapsulation of cells are familiar to theskilled artisan, and preparation of encapsulated cells and theirimplantation in patients may be accomplished without undueexperimentation. See, e.g., U.S. Pat. Nos. 4,892,538; 5,011,472; and5,106,627. A system for encapsulating living cells is described in PCTWO 91/10425 (Aebischer et al.). Techniques for formulating a variety ofother sustained or controlled delivery means, such as liposome carriers,bio-erodible particles or beads, are also known to those in the art, andare described, for example, in U.S. Pat. No. 5,653,975. The cells, withor without encapsulation, may be implanted into suitable body tissues ororgans of the patient.

As discussed above, it may be desirable to treat cell preparations withone or more CRP1 or B7RP1 polypeptides, variants, derivatives and/orfragments. This can be accomplished by exposing, for example, cellscomprising T-cells, such as bone marrow cells, to the polypeptide,variant, derivative, or fragment directly, where it is in a form that ispermeable to the cell membrane. For example, cells comprising T-cellsmay be exposed to a B7RP1 polypeptide in order to activate T-cellfunction and the cells so treated are implanted in the patient.

Alternatively, gene therapy can be employed. One manner in which genetherapy can be applied is to use a CRP1 or B7RP1 gene (either genomicDNA, cDNA, and/or synthetic DNA encoding a CRP1 or B7RP1 polypeptide, ora fragment, variant, or derivative thereof) which may be operably linkedto a constitutive or inducible promoter to form a “gene therapy DNAconstruct”. The promoter may be homologous or heterologous to theendogenous CRP1 or B7RP1 gene, provided that it is active in the cell ortissue type into which the construct will be inserted. Other componentsof the gene therapy DNA construct may optionally include, as required,DNA molecules designed for site-specific integration (e.g., endogenousflanking sequences useful for homologous recombination), tissue-specificpromoter, enhancer(s) or silencer(s), DNA molecules capable of providinga selective advantage over the parent cell, DNA molecules useful aslabels to identify transformed cells, negative selection systems, cellspecific binding agents (as, for example, for cell targeting)cell-specific internalization factors, and transcription factors toenhance expression by a vector as well as factors to enable vectormanufacture.

This gene therapy DNA construct can then be introduced into thepatient's cells (either ex vivo or in vivo). One means for introducingthe gene therapy DNA construct is via viral vectors. Suitable viralvectors typically used in gene therapy for delivery of gene therapy DNAconstructs include, without limitation, adenovirus, adeno-associatedvirus, herpes simplex virus, lentivirus, papilloma virus, and retrovirusvectors. Some of these vectors, such as retroviral vectors, will deliverthe gene therapy DNA construct to the chromosomal DNA of the patient'scells, and the gene therapy DNA construct can integrate into thechromosomal DNA; other vectors will function as episomes and the genetherapy DNA construct will remain in the cytoplasm. The use of genetherapy vectors is described, for example, in U.S. Pat. Nos. 5,672,344,5,399,346.

Alternative means to deliver gene therapy DNA constructs to a patient'scells without the use of viral vectors include, without limitation,liposome-mediated transfer, direct injection of naked DNA,receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,“gene gun”). See U.S. Pat. No. 4,970,154, WO 96/40958, U.S. Pat. Nos.5,679,559, 5,676,954, and 5,593,875.

Another means to increase endogenous CRP1 or B7RP1 polypeptideexpression in a cell via gene therapy is to insert one or more enhancerelements into the CRP1 or B7RP1 polypeptide promoter, where the enhancerelement(s) can serve to increase transcriptional activity of a CRP1 orB7RP1 polypeptide gene. The enhancer element(s) used will be selectedbased on the tissue in which one desires to activate the gene(s);enhancer elements known to confer promoter activation in that tissuewill be selected. For example, if a CRP1 or B7RP1 polypeptide is to be“turned on” in T-cells, the lck promoter enhancer element may be used.Here, the functional portion of the transcriptional element to be addedmay be inserted into a fragment of DNA containing a CRP1 or B7RP1polypeptide promoter (and optionally, vector, 5′ and/or 3′ flankingsequence, etc.) using standard cloning techniques. This construct, knownas a “homologous recombination construct” can then be introduced intothe desired cells either ex vivo or in vivo.

Gene therapy can be used to decrease CRP1 or B7RP1 polypeptideexpression by modifying the nucleotide sequence of the endogenouspromoter(s). Such modification is typically accomplished via homologousrecombination methods. For example, a DNA molecule containing all or aportion of the promoter of a CRP1 or B7RP1 gene(s) selected forinactivation can be engineered to remove and/or replace pieces of thepromoter that regulate transcription. Here, the TATA box and/or thebinding site of a transcriptional activator of the promoter may bedeleted using standard molecular biology techniques; such deletion caninhibit promoter activity thereby repressing transcription of thecorresponding CRP1 or B7RP1 gene. Deletion of the TATA box ortranscription activator binding site in the promoter may be accomplishedby generating a DNA construct comprising all or the relevant portion ofa CRP1 or B7RP1 polypeptide promoter(s) (from the same or a relatedspecies as a CRP1 or B7RP1 gene(s) to be regulated) in which one or moreof the TATA box and/or transcriptional activator binding sitenucleotides are mutated via substitution, deletion and/or insertion ofone or more nucleotides such that the TATA box and/or activator bindingsite has decreased activity or is rendered completely inactive. Thisconstruct, which also will typically contain at least about 500 bases ofDNA that correspond to the native (endogenous) 5′ and 3′ flankingregions of the promoter segment that has been modified, may beintroduced into the appropriate cells (either ex vivo or in vivo) eitherdirectly or via a viral vector as described above. Typically,integration of the construct into the genomic DNA of the cells will bevia homologous recombination, where the 5′ and 3′ flanking DNA sequencesin the promoter construct can serve to help integrate the modifiedpromoter region via hybridization to the endogenous chromosomal DNA.

Other gene therapy methods may also be employed where it is desirable toinhibit one or more CRP1 or B7RP1 polypeptides. For example, antisenseDNA or RNA molecules, which have a sequence that is complementary to atleast a portion of a selected CRP1 or B7RP1 polypeptide gene(s) can beintroduced into the cell. Typically, each such antisense molecule willbe complementary to the start site (5′ end) of each selected CRP1 orB7RP1 gene. When the antisense molecule then hybridizes to thecorresponding CRP1 or B7RP1 polypeptide mRNA, translation of this mRNAis prevented.

Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more CRP1 or B7RP1 polypeptides.In this situation, the DNA encoding a mutant full length or truncatedpolypeptide of each selected CRP1 or B7RP1 polypeptide can be preparedand introduced into the cells of a patient using either viral ornon-viral methods as described above. Each such mutant is typicallydesigned to compete with endogenous polypeptide in its biological role.

Agonists and Antagonists

The invention also provides for agonists and antagonists of CRP1 orB7RP1 which regulate the activity of either or both molecules. A CRP1 orB7RP1 agonist will stimulate or enhance at least one activity of CRP1 orB7RP1. A CRP1 or B7RP1 antagonist will partially or completely inhibitat least one activity of CRP1 or B7RP1. Agonists and antagonists may beidentified from test molecules which alter the binding of B7RP1 to CRP1.

The term “test molecule(s)” refers to the molecule(s) that is/are underevaluation for the ability to bind a CRP1 or B7RP1 polypeptide andthereby alter the binding of B7RP1 to CRP1. Preferably, the testmolecule will bind with an affinity constant of at least about 10⁶M.

A variety of assays may be used to measure binding of B7RP1 to CRP1.These assays may be used to screen test molecules for their ability toincrease or decrease the rate or extent of binding of B7RP1 to CRP1. Inone type of assay, a CRP1 polypeptide, preferably a soluble form of CRP1such as an extracellular domain, is immobilized by attachment to thebottom of the wells of a microtiter plate. Radiolabeled B7RP1 and thetest molecule(s) can then be added either one at a time (in eitherorder) or simultaneously to the wells. After incubation, the wells canbe washed and counted using a scintillation counter for radioactivity todetermine the extent of binding to CRP1 protein by B7RP1. Typically, themolecules will be tested over a range of concentrations, and a series ofcontrol wells lacking one or more elements of the test assays can beused for accuracy in evaluation of the results. An alternative to thismethod involves reversing the “positions” of the proteins, i.e.,immobilizing B7RP1 to the microtiter plate wells, incubating with thetest molecule and radiolabeled CRP1, and determining the extent of CRP1binding (see, for example, chapter 18 of Current Protocols in MolecularBiology, Ausubel et al., eds., John Wiley & Sons, New York, N.Y.[1995]).

As an alternative to radiolabelling, CRP1 or B7RP1 may be conjugated tobiotin and the presence of biotinylated protein can then be detectedusing streptavidin linked to an enzyme, such as horse radish peroxidase[HRP] or alkaline phosphatase [AP], that can be detectedcolorometrically, or by fluorescent tagging of streptavidin. An antibodydirected to CRP1 or B7RP1 that is conjugated to biotin may also be usedand can be detected after incubation with enzyme-linked streptavidinlinked to AP or HRP

CRP1 and B7RP1 may also be immobilized by attachment to agarose beads,acrylic beads or other types of such inert substrates. Thesubstrate-protein complex can be placed in a solution containing thecomplementary protein and the test compound; after incubation, the beadscan be precipitated by centrifugation, and the amount of binding betweenCRP1 and B7RP1 can be assessed using the methods described above.Alternatively, the substrate-protein complex can be immobilized in acolumn and the test molecule and complementary protein passed over thecolumn. Formation of a complex between CRP1 and B7RP1 can then beassessed using any of the techniques set forth above, i.e.,radiolabelling, antibody binding, or the like.

Another type of in vitro assay that is useful for identifying a testmolecule which increases or decreases formation of an CRP1/B7RP1 complexis a surface plasmon resonance detector system such as the Biacore assaysystem (Pharmacia, Piscataway, N.J.). The Biacore system may be carriedout using the manufacturer's protocol. This assay essentially involvescovalent binding of either CRP1 or B7RP1 to a dextran-coated sensor chipwhich is located in a detector. The test compound and the othercomplementary protein can then be injected into the chamber containingthe sensor chip either simultaneously or sequentially and the amount ofcomplementary protein that binds can be assessed based on the change inmolecular mass which is physically associated with the dextran-coatedside of the of the sensor chip; the change in molecular mass can bemeasured by the detector system.

In some cases, it may be desirable to evaluate two or more testcompounds together for use in increasing or decreasing formation of aCRP1/B7RP1 complex. In these cases, the assays set forth above can bereadily modified by adding such additional test compound(s) eithersimultaneously with, or subsequently to, the first test compound. Theremainder of steps in the assay are as set forth above.

In vitro assays such as those described above may be used advantageouslyto screen rapidly large numbers of compounds for effects on complexformation by CRP1 and B7RP1. The assays may be automated to screencompounds generated in phage display, synthetic peptide and chemicalsynthesis libraries.

Compounds which increase or decrease complex formation of CRP1 and B7RP1may also be screened in cell culture using cells and cell linesexpressing either polypeptide. Cells and cell lines may be obtained fromany mammal, but preferably will be from human or other primate, canine,or rodent sources. The binding of B7RP1 to cells expressing CRP1 on thesurface is evaluated in the presence or absence of test molecules andthe extent of binding may be determined by, for example, flow cytometryusing a biotinylated antibody to B7RP1. Cell culture assays may be usedadvantageously to further evaluate compounds that score positive inprotein binding assays described above.

Therapeutic Uses

Polypeptides of the invention, and agonists and antagonists thereof, maybe used to regulate T-cell function. Agonists and antagonists includethose molecules which regulate CRP1 and/or B7RP1 activity and eitherincrease or decrease at least one activity of a CRP1 or B7RP1 proteinsuch as one activity associated with T-cell functions, for example,T-cell activation. Agonists or antagonists may be co-factors, such as aprotein, peptide, carbohydrate, lipid, or small molecular weightmolecule, which interact with either CRP1 or B7RP1 and thereby regulatetheir activity. Potential polypeptide agonists or antagonists includeantibodies that react with either soluble or membrane-bound forms ofCRP1 or B7RP1 which comprise part or all of the extracellular domains ofthe said proteins. Molecules that regulate CRP1 or B7RP1 expressiontypically include nucleic acids encoding CRP1 or B7RP1 protein that canact as anti-sense regulators of expression.

CRP1 or B7RP1 polypeptides, and agonists and antagonists thereof, may beused in the treatment of autoimmune disease, graft survival, immune cellactivation for inhibiting tumor cell growth, T-cell dependent B-cellmediated diseases, and cancer gene immunotherapy. In one embodiment,antagonists or inhibitors of CRP1 and/or B7RP1 function may bebeneficial to alleviate symptoms in diseases with chronic immune celldysfunction. Autoimmune diseases, such as systemic lupus erythematosis,rheumatoid arthritis, immune thrombocytopenic purpura (ITP), andpsoriasis, may be treated with antagonists or inhibitors of CRP1/B7RP1.In addition, chronic inflammatory diseases, such as inflammatory boweldisease (Crohn's disease and ulcerative colitis), Grave's disease,Hashimoto's thyroiditis, and diabetes mellitis, may also be treated withinhibitors to CRP1/B7RP1. As described in Example 18, CRP1-Fc inhibitsand B7RP1-Fc enhances, the onset of disease in a rodent rheumatoidarthritis disease model. These opposite effects in this model support anagonistic role for the B7RP1-Fc protein and an antagonistic role for theCRP1-Fc protein. The results also illustrate how T-cell responses can beregulated by manipulation of this pathway and the significance of thispathway in the progression of rheumatoid arthritis. In addition, asdescribed in Example 19, expression of B7RP1-Fc in vivo stimulates aninflammatory bowel disease (IBD) phenotype in transgenic mice. Thisexample supports the role for B7RP1/CRP1 in the development ofinflammation in the intestine. Therefore, antagonists of the B7RP1/CRP1pathway may be used to treat human IBD.

Antagonists of CRP1 or B7RP1 may be used as immunosuppressive agents forbone marrow and organ transplantation and may be used to prolong graftsurvival. Such antagonists may provide significant advantages overexisting treatment. Bone marrow and organ transplantation therapy mustcontend with T-cell mediated rejection of the foreign cells or tissue bythe host. Present therapeutic regimens for inhibiting T-cell mediatedrejection involve treatment with the drugs cyclosporine or FK506. Whiledrugs are effective, patients suffer from serious side effects,including hepatotoxicity, nephrotoxicity, and neurotoxicity. The targetfor the cyclosporin/FK506 class of therapeutics is calcineurin, aphosphatase with ubiquitous expression. Since CRP1 expression isrestricted to T-cells, inhibitors of CRP1 or B7RP1 may lack the severeside effects observed with the use of the present immunotherapeuticagents.

Antagonists of CRP1 or B7RP1 may be used as immunosuppressive agents forautoimmune disorders, such as rheumatoid arthritis, psoriasis, multiplesclerosis, diabetes, and systemic lupus erythematosus.

Antagonists of the CRP1/B7RP1-mediated costimulatory pathway may also beused to alleviate toxic shock syndrome, inflammatory bowel disease,allosensitization due to blood transfusions, T-cell dependent B-cellmediated diseases, and the treatment of graft vs. host disease.

Antibodies, soluble proteins comprising for example extracellulardomains, and other regulators of CRP1 or B7RP1 that result in prolongedor enhanced T-cell activation can be used to increased the immuneresponse to tumors. Example 20 shows B7RP1-Fc can inhibit tumor cellgrowth in mice. Similarly, human B7RP1-Fc, or other activators of theB7RP1/CRP1 pathway, may be used to enhance immune responses againsthuman tumors. Anti-tumor activity is generally considered to have astrong cytolytic T-lymphocyte component. In fact, the anti-tumor effectsof B7-Fc fusion proteins (Sturmhoefel et al., Cancer Res. 59: 4964-4972,1999) were mediated by cytolytic CD8+ T-cells. Since CRP1 is alsoexpressed on cytolytic CD8+ T-cells (Example 9), it is probable that theanti-tumor effects demonstrated in Example 20 were due to B7RP1-Fcaction on CD8+ cells. The B7RP1/CRP1 pathway can also be manipulated toregulate CTL response in a number of other clinical settings, includingallograft transplantation, graft vs. host disease, and autoimmunediseases.

Gene therapy using B7RP1 genes of the invention may be used in cancerimmunotherapy. B7RP1 genes introduced into cancer cells can transformthem into antigen presenting cells that can be recognized by the T-cellsof the immune system when introduced back into an animal. Recognition ofthe transfected tumor cells by the T-cells results in eradication ofboth tumors cells expressing, or not expressing, the B7RP1 gene. Thisimmunotherapy approach may be used for various leukemias, sarcomas,melanomas, adenocarcinomas, breast carcinomas, prostate tumors, lungcarcinomas, colon carcinomas and other tumors. This inventionencompasses using the B7RP1 gene in a similar manner to enhance T-cellactivation in response to variety of tumors.

As described in Example 14, the phenotype of transgenic mice expressingB7RP1 indicates that B7RP1 is important in the control of antibodyproduction. Agonists and antagonists of B7RP1 protein activity may beuseful in therapeutic indications that call for the inhibition orenhancement of antibody production.

For instance, many vaccines act by eliciting an effective and specificantibody response. Some vaccines, especially those against intestinalmicro-organisms (e.g. Hepatitis A virus, and Salmonellas), elicit ashort-lived antibody response. It is desirable to potentiate and prolongthis response in order to increase the effectiveness of the vaccine.Therefore, soluble B7RP1 or activating antibodies to CRP1 may serve as avaccine adjuvant.

Anti-viral responses may also be enhanced by activators or agonists ofthe B7RP1/CRP1 pathway. The data in Example 20 indicate that cellularimmunity is enhanced by B7RP1-Fc. The enhancement of cellular immunefunctions by B7RP1-Fc, or other activators of the B7RP1/CRP1 pathway,may also be beneficial in eliminating virus-infected cells. In acomplementary fashion, B7RP1-Fc has effects on humoral immune functionsthat may enhance antibody mediated responses as observed in Example 13that may function to help clear free-virus from the body.

Enhancement of cellular immune functions, would be desirable in treatingcancer or viral infection. Immune response may be enhanced by activationof the B7RP1/CRP1 pathway optionally in conjunction with activation of aseparate immune stimulating pathway. A synergistic effect of B7RP1 andB7.2 in a mouse chronic hypersensitivity model is shown in Example 24.Thus, B7RP1 or an agonist of CRP1 may be administered with B7.1 or B7.2to stimulate immune functions. In another embodiment, B7RP1 or anagonist of CRP1 may be administered with a CD28 agonist or a CTLA4antagonist to stimulate immune functions. It is also contemplated thatB7RP1 or a CRP1 agonist may be used with other immune stimulatingmolecules, such as T-cell stimulating molecules, in order to enhancecellular immune functions. In one embodiment, a CRP1 agonist is anantibody which binds CRP1 and stimulates or enhances CRP1 activity.

Conversely, there are a number of clinical conditions that would beameliorated by the inhibition of antibody production. Hypersensitivityis a normally beneficial immune response that is exaggerated orinappropriate, and leads to inflammatory reactions and tissue damage.Hypersensitivity reactions which are antibody-mediated may beparticularly susceptible to antagonism by inhibitors of B7RP1 activity.Allergies, hay fever, asthma, and acute edema cause type Ihypersensitivity reactions, and these reactions may be suppressed byprotein, antibody or small molecule inhibitors of B7RP1 activity.

Diseases that cause antibody-mediated hypersensitivity reactions,including systemic lupus erythematosis, arthritis (rheumatoid arthritis,reactive arthritis, psoriatic arthritis), nephropathies(glomerulo-nephritis, membranous, mesangiocapillary, focal segmental,focal necrotizing, crescentic, proliferative-tubulopathies), skindisorders (pemphigus and pemphigoid, erythema nodosum), endocrinopathies(thyroiditis-Grave's, Hashimoto's-insulin dependent diabetes mellitus),various pneumopathies (especially extrinsic alveolitis), variousvasculopathies, coeliac disease, with aberrant production of IgA, manyanemias and thrombocytopenias, Guillain-Barre Syndrome, and myastheniagravis, may be treated with B7RP1 antagonists.

In addition, lymphoproliferative disorders, such as multiple myeloma,Waldenstrom's macroglobulinemia, and crioglobulinemias, may be inhibitedby protein, antibody, or small molecule antagonists of B7RP1.

Finally, graft versus host disease, an “artificial” immune disorder, maybenefit from the inhibition of antibody production by B7RP1 antagonists.

The B7RP1/CRP1 pathway is involved in regulating IgE production. IgE isan immunoglobulin isotype specifically involved in mediating allergicresponses such as asthma, food allergies, hay fever, type 1hypersensitivity and sinus inflammation. Upon exposure to an allergen, aprocess involving T-cell and B cell collaboration results in B cellproduction of IgE specific for the allergen. Allergen-specific IgEreleased into the circulation by B cells bind to mast cells andbasophils through the high affinity IgE receptor (Fc_(ε)RI). Mast cellsand basophils to which IgE is bound become sensitized and subsequentexposure to the allergen results in cross-linking of the surfacereceptors and release of histamines.

Example 22 shows that transgenic mice expressing a B7RP1 fusion proteinhave increased levels of IgE compared to normal mice. In addition, CRP1“knockout” mice show complete inhibition of class switching to IgE (seeExample 23). These results indicate that activation of the B7RP1/CRP1pathway leads to IgE production.

The invention provides for use of modulators of B7RP1 or CRP1 toregulate IgE production and to prevent or treat IgE-mediated disorders.In one embodiment, antagonists of B7RP1 or CRP1 are used to partially orcompletely inhibiting IgE production. The antagonists may be usedseparately, or in combination, in a treatment regimen for decreasing IgElevels. Examples of B7RP1 and CRP1 antagonists include nucleic acids,polypeptides, peptides, antibodies, carbohydrates, lipids, and smallmolecules. In one embodiment, the antagonist is an antibody which bindsto B7RP1 and partially or completely inhibits IgE production. In anotherembodiment, the antagonist is an antibody which binds to CRP1 andpartially or completely inhibits CRP1 activity. In another embodiment, acombination of a B7RP1 antagonist and a CRP1 antagonist may be used, forexample, B7RP1 antagonist antibody and a CRP1 antagonist antibody. B7RP1and CRP1 antagonists are administered in amounts effective to decreaseIgE production.

The antagonists of the invention may be used to prevent and/or treatdisorders characterized by excessive or inappropriate IgE production. Byway of example, such disorders include allergic responses such asasthma, food allergies, hay fever, hypersensivity, and sinusinflammation.

The invention also provides for the use of a B7RP1 antagonist or a CRP1antagonist in combination with an IgE antagonist to partially orcompletely inhibit IgE production and to prevent and/or treat disorderscharacterized by excessive or inappropriate IgE production. As usedherein the term “IgE antagonist” refers to a compound capable ofdisrupting blocking the interaction of IgE with its high affinityreceptor Fc_(ε)RI on cells such that the response to allergen stimulusis attenuated or eliminated. Antagonists include an anti-IgE antibodyand fragments thereof, soluble Fc_(ε)RI receptor and fragments thereof,anti-Fc_(ε)RI antibody and fragments thereof, IgE variants and fragmentsthereof, IgE binding peptides, Fc_(ε)RI receptor binding peptides, andsmall molecules capable of binding to IgE or competing with IgE forbinding to Fc_(ε)RI receptor. B7RP1 antagonists may also be used with incombination with antihistamines, allergen desensitization, reduction inexposure to allergen and the like for treatment of allergic disorders.

In some instances, it may be useful to increase IgE production, such asto prevent and/or treat immune-related disorders in an immune comprisedhost. In such cases, B7RP1 alone or in conjunction with a CRP1 agonistmay be administered in amounts sufficient to increase IgE production.

The invention also provides for the prevention and/or treatment ofasthma comprising administering a B7RP1 antagonist or a CRP1 antagonistalone or in conjunction with one or more agents for treating asthma.Examples of such agents include bronchodilators (anti-cholinergicagents, β₂ adrenergic receptor agonists, lenkotriene D₄ antagonists,neurokinin antagonists, potassium channel openers, substance Pantagonists, thromboxane A₂ antagonists, and xanthines),anti-inflammatories (5-lipoxygenase inhibitors, 5-lipoxygenaseactivating protein inhibitors, phosphodiesterase IV inhibitors, plateletactivating factor antagonists, respiratory NSAIDS, steroids, andtyrosine kinase inhibitors), cytokine inhibitors (CD4, IL-4 and IL-5inhibitors) and IgE antagonists as set forth above.

The following examples are offered to more fully illustrate theinvention, but are not construed as limiting the scope thereof.

Example 1 CRP1 cDNA and Amino Acid Sequence

Female C57/Black 6 mice were sacrificed, and the small intestines wereexcised, and the Peyer's patches were removed. The small intestinetissue was sliced open and washed to remove mucus and other debris. Theepithelial layer, which contains the intestinal intraepithelial cells(iIELs), was released by gentle agitation in RPMI-1640 supplemented with1 mM dithiothreitol (DTT), for 20 minutes at 37° C. Disassociated cellswere passed through a 100μ filter, washed in 50 ml of RPMI-1640, mixedto further break up clumps of cells, and then passed through a 40μstrainer to obtain single cell populations. These cells were then washedagain in a 50 ml volume of RPMI-1640 to ensure the removal of theresidual DTT. The tissue was then agitated and washed as before togather the remaining iIELs. The iIELs were separated from the adiposecells and most epithelial cells on a 3-step Percol gradient, with theiIELs banding at the 40% to 80% interface. These cells were then washedtwice with RPMI-1640 to remove traces of Percol, immunostained withCD103 (integrin alpha IEL) antibodies, and separated on a FACS Star cellsorter. These sorted cells were then either used to prepare total RNAdirectly using Trizol (Gibco BRL, Gaithersburg, Md.), or activatedovernight on plate-bound activating antibodies, which crosslink thegamma/delta TCR, alpha/beta TCR, or CD3. The RNA was prepared as aboveand pooled for use in constructing EST cDNA libraries.

A cDNA clone, designated smil2-00082-al, contained nucleotide sequencehomology to CD28 (FIG. 1B). Translation of the sequence and subsequentcomparison to known proteins in a public database revealed 19% aminoacid identity with murine CD28 (FIG. 1B). This low homology wassignificant because murine CD28 shares only 26% amino acid identity withmurine CTLA-4. All of the putative cysteines thought to be critical forintra- and inter-molecular cysteine bonding in the CD28/CTLA-4 familywere found to be conserved (amino acid residues 83, 109, and 137;relative to the initiating methionine). In addition, the overall lengthof the putative open reading frame, and the relative position of thetransmembrane domain, were similar to those of both CD28 and CTLA-4. Wenamed the gene CRP1, for CD28-Related Protein-1.

Example 2 Cloning of Human CRP1 cDNA

The nucleic acid sequence encoding human CRP1 protein is identified bythe following procedures. A human cDNA library was prepared fromenriched lymphocytes from peripheral human blood from normal humanvolunteers. The lymphocytes were purified and red blood cells wereremoved by Lymphocyte Separation Media (ICN Pharmaceuticals, Inc., CostaMesa, Calif.). The cells were then activated overnight in mediacontaining 10 ng/ml PMA, 500 ng/ml ionomycin, and plate-bound activatingantibodies to CD3. Total RNA was prepared from the activated cells bythe Trizol method (Gibco/BRL) and poly A RNA was isolated by Dynal beadpurification. cDNA was made from the isolated poly A RNA and sizeselected for largest cDNA fragments. The size selected cDNA was thenligated into the plasmid pSPORT (Gibco/BRL). DNA encoding human CRP1protein is obtained by screening the activated lymphocyte cDNA libraryby either recombinant bacteriophage plaque, or transformed bacteriacolony hybridization protocols (Sambrook et al. Supra). The phage orplasmid cDNA library are screened using radioactively-labeled probesderived from the murine CRP1 gene clone as described in Example 1 andFIG. 1. The probes are used to screen nylon filters lifted from theplated library. These filters are prehybridized for 4 hr at 42° C. in50% formamide, 5×SSPE, 2×Denhardt's solution, 0.5% SDS, and 100 μg/mlsalmon sperm DNA and then hybridized for 24 hr at 42° C. in 50%formamide, 5×SSPE, 2×Denhardt's solution, 0.5% SDS, 100 μg/ml salmonsperm DNA, and 5 ng/ml mB7RP1 probe. The blots are washed in 2×SSC, 0.1%SDS for 10 min at RT, 1×SSC, 0.1% SDS for 10 min at 50° C., 0.2×SSC,0.1% SDS for 10 min at 50° C., then 0.2×SSC for 10 min at 50° C. again.Inserts obtained from any human CRP1 clones are sequenced and analyzedas described in Example 1.

Example 3 B7RP1 DNA and Amino Acid Sequence

A cDNA clone, designated smil1-00003-g5, contained nucleotide sequencehomology to B7.1 (CD80) and B7.2 (CD86). Translation of the sequence(FIG. 2A) and subsequent comparison to known proteins in a publicdatabase revealed 20% amino acid identity with murine B7.1 (FIG. 2B).This low homology was significant because murine B7.1 shares only 24%amino acid identity with murine B7.2. Despite this low homology,critical cysteine residues are conserved between the open reading frameof this clone and murine B7.1 and B7.2 at residues 62, 138, 185, and 242(relative to the initiating methionine, FIG. 2B). The approximate matureprotein length and the location of the transmembrane region relative tothe carboxy terminus are also similar in the putative ORF of this clone,as compared to B7.1 and B7.2. We named the gene B7RP1, for B7-RelatedProtein-1.

Example 4 Cloning of Human B7RP1 cDNA

A Genbank blast homology search (GCG, University of Wisconsin) usingmurine B7RP1 sequence (see FIG. 2) retrieved a clone (AB014553)containing a 4358 bp sequence with 1679 bp of ORF. PCR cloning primerswere designed according to this sequence. A DNA fragment of 1313 bp wasobtained by 5′ and 3′ RACE using Human Lymph Node Marathon-Ready™ cDNA(Clontech, Palo Alto, Calif.) according to the manufacturer'srecommended procedures.

Primers used for full length human B7RP1:

(SEQ ID NO: 25) 2083-75 ACC ATG CGG CTG GGC AGT CCT GGA (SEQ ID NO: 26)2083-76 TGG TGA CCT ACC ACA TCC CAC AG (SEQ ID NO: 27) 2083-77TCC GAT GTC ATT TCC TGT CTG GC (SEQ ID NO: 28) 2083-78GCT CTG TCT CCG GAC TCA CAG CCC (SEQ ID NO: 29) 2113-29GTG GCA GCA AAC TTC AGC GTG CCC GTC G (SEQ ID NO: 30) 2113-30CCC AAC GTG TAC TGG ATC AAT AAG ACG G (SEQ ID NO: 31) 2113-31GCG TGC TGA GGA TCG CAC GGA CCC CCA GPrimers 2083-75 and 2083-76 were used to amplify the 5′ end of the geneusing RACE protocols. Primers 2083-77, 2083-78, 2113-29, 2113-30, and2113-31, were used to amplify the 3′ end of the gene using RACEprotocols.

The resulting nucleotide sequence contained an ORF of 288 amino acidresidues beginning at the methionine. The predicted mature human B7RP1amino acid sequence was then compared to the mature mouse B7RP1 aminoacid sequence (FIG. 3B) and found to share 48% amino acid identity. Thishomology is significant because the homology between species is low withthe CD80 (B7.1) gene, in fact, the mouse and human CD80 share only 41%amino acid identity. Importantly, the human B7RP1 protein conservecritical cysteine residues necessary for Ig loop structures (amino acidresidues 16, 92, 138, 194, and 195, relative to the mature protein, FIG.3B). In addition, the overall length and position of the transmembranedomain are consistent with a human B7RP1 homolog.

Example 5 Expression of B7RP1 RNA

RNA in situ hybridization using RNA probes to the B7RP1 gene. Adultmouse tissues were fixed in 4% paraformaldehyde, embedded in paraffin,and sectioned at 5 μm. Prior to in situ hybridization, tissues werepermeabilized with 0.2M HCL, followed by digestion with Proteinase K,and acetylation with triethanolamine and acetic anhydride. Sections werehybridized overnight at 55° C. with a 969 base ³³P-labeled riboprobecorresponding to nucleotides 1 to 969 of the mouse B7RP1 sequence.Excess probe was removed by RNase digestion, followed by a series ofwashes in buffer with decreasing salt concentrations, and then a highstringency wash in 0.1×SSC at 55° C. Slides were dipped in Kodak NTB2emulsion, exposed at 4° C. for 2-3 weeks, developed, and counterstainedwith hematoxilyn and eosin. Sections were examined with darkfield andtransmitted light illumination to allow simultaneous evaluation of thetissue morphology and the hybridization signal.

The analysis of the B7RP1 RNA by in situ hybridization showed that theB7RP1 RNA was highly expressed in areas of lymphoid maturation andlymphocyte activation. B7RP1 RNA was expressed in the lymphoid tissuesof the thymus, Peyer's patches of the intestine, spleen, and lymphnodes. Expression within these lymphoid tissues demonstrated that theB7RP1 RNA was generally expressed in the areas of B-cell and other APCinvolvement. These regions include the medulla area of the thymus, theprimary follicles of the lymph nodes, and the follicular and domeregions of the Peyer's patches. The expression of B7RP1 RNA is highlyspecific to the regions of APC involvement in lymphoid tissues.

The analysis of several non-lymphoid tissues also revealed B7RP1expression in regions of APC involvement. In the lung, B7RP1 expressionwas found in the submucosal regions, consistent with a function inantigen processing. In the small intestine, B7RP1 RNA was found in thelamina propria. Notably, we found a section of damaged liver, whichshowed lymphocyte infiltration that overlapped with the expression ofB7RP1 RNA. This coincidence of B7RP1 expression with lymphocyteaccumulation in response to tissue damage strongly indicates that B7RP1is involved in lymphocyte activation.

Example 6 Expression of CRP1 RNA

RNA in situ hybridization using RNA probes to the CRP1 gene. Mousetissues were prepared as in Example 5. Tissue permeabilization, probehybridization, slide treatment, and tissue staining were as described inExample 5. Sections were hybridized overnight at 55° C. with a 603 base³³P-labeled riboprobe corresponding to nucleotides 1 to 603 of the mouseCRP1 sequence. Sections were examined with darkfield and transmittedlight illumination to allow simultaneous evaluation of the tissuemorphology and the hybridization signal.

Lymph nodes from normal mice or a mouse treated with oxazolone weresectioned and analyzed for CRP1 RNA expression. The sensitized mouselymph node showed greater expression of CRP1 RNA than the normal mouselymph node. The expression of CRP1 was in the paracortex, a region ofT-cell activity. Therefore, the expression of CRP1 RNA is consistentwith that of T-lymphocyte expression and is up-regulated upon T-cellactivation.

Example 7 Expression and Purification of CRP1-Fc and B7RP1-Fc FusionProteins

To construct the DNA expression vector for the CRP1-Fc fusion protein,the coding sequence for the first amino terminal 147 amino acids of theCRP1 was fused, inframe, to the coding sequence for the carboxy terminal235 amino acids of the human Fc gene (isotype IgG1) and ligated withinthe polylinker sequence of pcDNA3 (pcDNA3/CRP1-Fc). To construct the DNAexpression vector for the B7RP1-Fc fusion protein, the coding sequencefor the first amino terminal 269 amino acids of the B7RP1 was fused,inframe, to the coding sequence for the carboxy terminal 235 amino acidsof the human Fc gene (isotype IgG1) and ligated within the polylinkersequence of pcDNA3 (pcDNA3/B7RP1-Fc). The coding sequences of both CRP1and B7RP1 contained sequences from the N-terminus of each protein up to,but not including, the putative transmembrane region of each protein.293T-cells were transfected with either pcDNA3/CRP1-Fc orpcDNA3/B7RP1-Fc using the FuGene 6 transfection reagent (Roche MolecularBiochemicals, Indianapolis, Ind.). After four days, the conditionedmedia were collected and the Fc fusion proteins were purified by batchchromatography using Protein A Sepharose (Pharmacia). Fc fusion proteinsbound to the column were eluted with three column volumes of ImmunopureGentle Elution Buffer (Pierce), and then were dialyzed against 150volumes of 20 mM HEPES, 100 mM NaCl, pH 7.5. The dialyzed protein wasconcentrated using Macrosep centrifugal concentrates, 30 kD MWCO (PallFiltron), and the protein concentrations were calculated usingextinction coefficients derived from the amino acid sequence of eachprotein. Expression of CRP1-Fc fusion protein is shown in FIG. 4A,expression of B7CPR1-Fc fusion protein is shown in FIG. 4B.

Example 8 Identification of CRP1 and B7RP1 as a Receptor-Ligand Pair

In order to determine whether the novel proteins were part of the samecostimulatory pathway as that containing CD28, CTLA-4, B7.1, and B7.2,we utilized a cell surface display assay. This assay uses ACAS (AdherentCell Analysis and Sorting) analysis to analyze whether membrane-boundproteins expressed in cells interact with various Fc fusion proteins.Cells expressing membrane-bound proteins, indicated on the left side ofFIG. 5, were incubated with Fc fusions proteins, indicated at the top ofthe figure.

Cos-7 cells, grown in DMEM media with 10% FBS, were plated at 500,000cells/well in a 24-well plate. Cells were transfected using the FuGene 6reagent (Roche Molecular Biochemicals, Indianapolis, Ind.). For eachtransfection, 3 μl of FuGene 6 reagent was added to 47 μl of serum freeDMEM media. After a 10 min incubation at room temperature, the mix wasadded to 0.25 μg of plasmid dropwise and then was incubated for 15minutes. The above mix was then added to the cells with 0.5 ml of DMEMwith 10% FBS. The cells were incubated at 37° C. in a 5% CO2 atmosphere.As a control, CHO D-cells, stably transfected with an expression plasmidcontaining the cDNA for human CD28, were also plated at 500,000cells/well in a 24-well plate.

After 48 hr, the medium with transfection reagent was removed and thecells were washed twice with RPMI plus 5% FBS. 10 to 20 ng of purifiedFc fusion proteins in 1 ml of media were added to the cells, which wereincubated for 30 min on ice. The cells were washed three times with RPMIplus 5% FBS and then were incubated with 2 μl of FITC-conjugatedanti-human Fc antibody (1 mg/ml) for another 30 min on ice. After threesuccessive washes with RPMI, the cells were covered with 250 μl of RPMImedia without phenol red for ACAS analysis.

ACAS analysis of the cells that bound the various Fc fusion proteinsdemonstrated that the B7RP1 protein bound CRP1, but not the proteins inthe known costimulatory pathway, CD28 or CTLA-4. Conversely, CRP1interacted with B7RP1, but not B7.2, a component in the known pathway.(See FIG. 5). These results strongly indicate that CRP1 and B7RP1represent a novel receptor-ligand pair, analogous to CD28 and B7.2.However, since CRP1 and B7RP1 do not interact with B7.2, CTLA-4, orCD28, they are separate and independent of the known costimulatorypathway.

Example 9 Identification of Cells Expressing B7RP1 Receptors

The B7RP1-Fc fusion protein was utilized to detect cells that expressedreceptors to B7RP1, presumably including the CRP1 protein (see Example6), by FACS analysis. Spleens were removed from female C57/Black 6 mice,ground on 100 micron mesh filters to release the lymphocytes, passedthrough 70 micron filters, and then washed in 50 ml of RPMI-1640. Theywere pelleted at 1500 rpm, resuspended in fresh RPMI, mixed to break upthe clumping cells, and passed through a 40 micron filter. T-cells to beactivated were seeded into 6 well plates in RPMI-1640, 5% FBS, 1×PSG,PMA, ionomycin, and incubated at 37° C., 5% CO2 overnight. T-cellactivation was checked by visual confirmation after 12 hr.

Activated spleen cells for immunostaining were washed in PBS, 0.5% BSA(Path-ocyte 4, ICN Pharmaceuticals) wash buffer, resuspended, and thenaliquoted in 100 μl volumes. 15 μg/ml of either the CRP1-Fc fusionprotein or the B7RP1-Fc fusion protein was added (1.5 μg/sample) asappropriate, and then the mixtures were incubated on ice for 30 min withoccasional mixing. The cells were washed twice in 5.0 ml of wash buffer.Binding of the fusion proteins was visualized with 2 μg ofgoat-anti-human (GaHuFc-FITC) conjugated secondary antibody in a 100 μlvolume for cell staining. Cell marker antibodies conjugated with PE wereadded with the GaHUFc-FITC, as well as control isotype-PE conjugatedantibody controls where indicated (rat isotype). The samples wereincubated on ice and washed as before. Visualization was done by FACScananalysis with gating on the lymphocyte populations. Double staining withCD4+ antibodies and the B7RP1-Fc fusion protein indicated that the cellsexpressed both the CD4 marker and the receptor to B7RP1, presumably CRP1(FIG. 6). Similarly, double staining with CD8+ antibodies and theB7RP1-Fc fusion protein demonstrated that cells expressed both CD8 andB7RP1 receptors (FIG. 6). We could not reliably detect such doublestaining cells in inactivated splenocyte preparations. Since CD4 and CD8are T-lymphocyte markers, we can postulate that CRP1 is expressed onactivated CD4+ and CD8+ T-cells. These data are consistent with theincreased expression of CRP1 RNA in the T-cell regions of lymph nodesfrom sensitized mice as compared to normal mice (Example 6).

Example 10 Identification of Cells Expressing CRP1 Ligands

The CRP1-Fc fusion protein was utilized to detect cells that expressedligands to CRP1, presumably including the B7RP1 protein (see Example 8),by FACS analysis (FIG. 7). Splenocytes were prepared as in Example 8,except the 12 hr T-cell activation step was omitted and the cells weredirectly analyzed. Splenocytes were double stained with CD45R (B220)marker antibodies and the CRP1-Fc fusion protein. Cells were detectedthat expressed both the CD45R B-cell marker and the putative ligands forCRP1, presumably including B7RP1 (Example 8). Therefore, we concludethat B7RP1 is expressed on B-cells, a type of antigen-presenting cell.These data are consistent with the expression of B7RP1 RNA in B-cellregions of various lymphoid tissues (Example 5).

FACS analysis of the expression of B7RP1 on peritoneal macrophages (FIG.8). Peritoneal cells were collected by local lavage from a normal mouseand washed before being incubated with the CRP1-Fc fusion protein or theFc protein as a control or with the F4/80 monoclonal antibody (whichdetects an antigen specific for macrophages) or an irrelevant,isotype-matched control monoclonal antibody. Cells were then washedagain and incubated with goat-anti-human Fc-FITC conjugated antibody.After further washing, cells were assessed in a FACS analyzer for theirlight scattering and fluorescence staining properties. Peritoneal cellswere first distinguished in subsets on the ground of their lightscattering properties (FIG. 8A). Macrophages were identified in region 5(R5) because of their ability to strongly scatter light forward (FSC)and sideways (SSC) and because of their positive staining for the F4/80antigen, a marker for macrophages (FIG. 8B). Macrophages in region 6(R6) were singled out on the basis of their less intense staining forthe F4/80 antigen and found to be stained by the CRP1-Fc fusion protein(FIG. 8C). These data indicate that ligands for CRP1, possibly includingB7RP1, are expressed on macrophages, a professional antigen presentingcell. This is consistent with CRP1 and B7RP1 function in T-lymphocyteactivation.

Example 11 In vitro Inhibitory Activity of the B7RP1-Fc Fusion Proteinon ConA-Stimulated T-Lymphocytes

Mouse splenocytes were prepared as in Example 8 and enriched forT-lymphocytes by negative selection (R and D Systems, Inc., Minneapolis,Minn.)). 200,000 splenocytes were used in T-cell proliferation assays ina 96-well round-bottom plate. Cells were incubated for 1 hr with media(no adds), CRP1-Fc, B7RP1-Fc, or B7.2-Fc, fusion proteins as indicatedFIG. 9. Media (no adds), or Con A at various concentrations were addedas indicated in at the bottom of FIG. 9. The cells were then incubatedat 37° C. and 5% CO2. After 42 hr, cells were pulsed with 3H-thymidinefor 6 hr, harvested and incorporated radioactivity determined. AverageCPM and standard deviation from triplicate samples are represented inFIG. 9.

The Fc fusion proteins did not demonstrate significant T-cellstimulatory or inhibitory activity by themselves, however, in thepresence of 1 μg/ml and 3 μg/ml Con A, both the B7RP1-Fc and the knownB7.2-Fc fusion proteins showed significant inhibitory activity (FIG. 9).At high concentrations (10 μg/ml), Con A stimulation results in celldeath, presumably through over-activation of the T-cells. Addition ofeither B7RP1-Fc or B7.2-Fc, significantly protected the cells from thedetrimental effects of high concentrations of Con A. In both inhibitoryand protective functions, the effect by B7RP1-Fc protein was greaterthan B7.2-Fc protein on the Con A stimulated cells. These data indicatethat the B7RP1 protein functions to regulate T-cell proliferation.

Example 12 Systemic Delivery of B7RP1-Fc Fusion Protein in TransgenicMice

The B7RP1-Fc fusion protein described in Example 7 was subcloned into anApoE-liver specific expression vector (Simonet et al. J. Clin. Invest.94, 1310-1319 (1994) and PCT Application No. US94/11675). The codingregion was excised from pCEP4/B7RP1-Fc using the restriction enzymes,Spe I and Not I, and the fragment subcloned into the same sites in thepreviously mentioned ApoE-liver specific expression vector. Theresultant plasmid, HE-B7RP1-Fc, was sequenced through it's proteincoding region, and sequences flanking the coding region, to ensure itwas mutation free.

The plasmid was amplified and purified through two rounds of CsCldensity gradient centrifugation. The purified plasmid DNA was digestedwith the restriction enzymes, Cla I and Ase I, and the 1.5 kb transgeneinsert was purified by agarose gel electrophoresis. The purifiedfragment was diluted to a stock injection solution of 1 μg/ml in 5 mMTris, pH 7.4, and 0.2 mM EDTA. Single-cell embryos from BDF1×BDF1-bredmice were injected essentially as described (Brinster et al., Proc.Natl. Acad. Sci. USA 82, 4338 (1985)), except that injection needleswere beveled and siliconized before use. Embryos were cultured overnightin a CO2 incubator and 15 to 20 2-cell embryos were transferred to theoviducts of pseudopregnant CD1 female mice.

Following term pregnancy, 56 offspring were obtained from implantationon the microinjected embryos. The offspring were screened by PCRamplification of the integrated transgene in genomic DNA samples. Thetarget region for amplification was a 369 bp region of the human Apo Eintron which was included in the expression vector. The oligos used forPCR amplification were:

5′-GCC TCT AGA AAG AGC TGG GAC-3′ (SEQ ID NO: 32)5′-CGC CGT GTT CCA TTT ATG AGC-3′ (SEQ ID NO: 33)

The conditions for the PCR were: 94° C. for 2 min, 1 cycle; 94° C. for 1min, 63° C. for 20 sec, and 72° C. for 30 sec, 30 cycles. Of the 56original offspring, 7 were identified as PCR positive transgenic foundermice.

At 12 weeks of age, nine transgenic founders (mouse #1, 2, 4, 6, 8, 30,32, 33, 40) and five controls (mouse #5, 9, 10, 25, 28) were sacrificedfor necropsy and pathological analysis. Total cellular RNA was isolatedfrom the livers of the founder animals and negative control littermatesas described (McDonald et al. Meth. Enzymol. 152, 219 (1987)). Northernblot analysis was performed on these samples to assess the level oftransgene expression. Approximately 10 μg of total RNA from each animalwas resolved by agarose electrophoresis denaturing gels (Ogden et al.Meth. Enzymol. 152, 61 (1987)), then transferred to HYBOND-N nylonmembrane (Amersham), and probed with ³²P dCTP-labeled mB7RP1-Fc insertDNA. Hybridization was performed for 1 hr at 63° C. in ExpressHybSolution (Clonetech) and 2−4×10⁶ CPM of labeled probe/ml hybridizationbuffer. Following hybridization, blots were washed twice in 2×SSC, 0.1%SDS at room temperature for 5 min each, and then twice in 0.1×SSC, 0.1%SDS at 55° C. for 15-20 min each. Expression of the transgene in founderand control littermates was determined following autoradiography.

Northern blot analyses indicated that seven of the transgenic foundersexpressed detectable levels of the transgene RNA (mouse #1, 2, 6, 8, 32,33, and 40). The negative control mice and three founders (#4, 30, and31) did not express detectable levels of RNA. Since the B7RP1-Fc fusionprotein was determined to be secreted from mammalian cells in culture(FIG. 4B and Example 7), expression of the transgene mRNA should beindicative of the level of systemically delivered gene product.

Example 13 Biological Activity of B7RP1-Fc Fusion Protein

Seven of the transgenic mice (mouse #1, 2, 6, 8, 32, 33, and 40) andfive control littermates (#5, 9, 10, 25, and 28) were sacrificed fornecropsy and pathological analysis using the following procedures: Priorto euthanasia, all animals had their identification numbers verified,then were weighed, anesthetized and blood drawn. The blood was saved asboth serum and whole blood for a complete serum chemistry and hematologypanel. Radiography was performed just after terminal anesthesia bylethal CO2 inhalation, and prior to gross dissection. Tissues were thenremoved and fixed in 10% buffered Zn-formalin for histologicalexamination. The tissues collected included the liver, spleen, pancreas,stomach, duodenum, ileum, Peyer's patches, colon, kidney, reproductiveorgans, skin, mammary glands, bone, brain, heart, lung, thymus, trachea,esophagus, thyroid/parathyroid glands, jejunum, cecum, rectum, adrenalglands, white and brown fat, sciatic nerve, bone marrow, urinarybladder, and skeletal muscle. Prior to fixation, the whole organ weightswere determined for the liver, heart, stomach, kidney, adrenals, spleen,and thymus. After fixation, the tissues were processed into paraffinblocks, and 3 μm sections were obtained.

Immunohistochemistry for the B-lymphocyte marker, B220, and theT-lymphocyte marker, CD3, was performed. To detect B220 or CD3expression, formalin fixed, paraffin embedded, 4 μm sections weredeparaffinized and hydrated to deionized water. The sections werequenched with 3% hydrogen peroxide, blocked with Protein Block (Lipshaw,Pittsburgh, Pa.), and incubated in rat monoclonal antibody to B220(Pharmingen, San Diego, Calif.) or rabbit polyclonal antibody to CD3(Dako, Carpinteria, Calif.). The antibodies were detected bybiotinylated rabbit anti-rat or goat anti-rabbit immunoglobulins,peroxidase conjugated streptavidin, (BioGenex, San Ramon, Calif.) withDAB as chromagen (Biotek, Santa Barbara, Calif.). Sections werecounterstained with hemaoxylin.

In this study, normal clinical signs were reported during the in-lifephase of the study. The whole body radiographs of the transgenic micewere comparable to those of the control mice. The overall hematologicparameters of the transgenic mice were comparable to those of thenegative control group, although sporadic changes in individual micewere present: transgenic #8 and #40 had increased serum globulin levels(hyperglobulinemia) and #32 and #33 had globulin levels in the highnormal range accompanied by albumin levels in the low normal range,which is a pattern commonly seen with chronic antigenic stimulation ofthe immune system. Organ weights of the other transgenic mice were notsignificantly different from those of the control group.

The following histopathological changes were present in the transgenicmice: The mesenteric lymph nodes of the transgenic B7RP1-Fc mice weremoderately to markedly enlarged when compared to the control mice (FIG.10A-10D; FIG. 11A-11E). The cortex had prominent follicular hyperplasiaseen as enlarged secondary follicles (FIG. 10B-11B) with large germinalcenters containing mostly B220+ B cells (FIG. 11D) and a few scatteredCD3+ T cells (FIG. 11F). The paracortical (CD3+ T-cell) area was alsomoderately enlarged (FIGS. 11B-11F) and the medullary sinuses hadslightly increased numbers of fleshy macrophages (sinus histiocytosis).The most conspicuous change in the nodes was present in the medullarycords, which were mildly to markedly expanded by large numbers ofwell-differentiated plasma cells in the B7RP1-Fc transgenic mice (FIG.10D). In transgenic mouse #40, small numbers of scattered Russell bodies(i.e. plasma cells with prominent, large, round, intracytoplasmicvesicles containing immunoglobulins) were also found in the medullarycords (FIG. 10D). Interestingly, the other internal and peripheral lymphnodes (e.g. cervical, inguinal) had similar morphologic features ofreactive lymphoid hyperplasia suggestive of a systemic response. Thesefindings are consistent with a chronic, ongoing immune stimulation withenhancement of the humoral immune reaction, which leads to B cellproliferation and terminal differentiation into plasma cells.

The spleen of B7RP1-Fc transgenic mice had variably enlarged white pulpareas with moderate reactive lymphoid hyperplasia involving particularlythe B-cell secondary follicles with prominent germinal centers andperiarteriolar T-cell sheaths when compared to the control mice (FIG.10E-10F). Another conspicuous finding in B7RP1-Fc transgenic mice wasminimal to mild plasmacytosis in the marginal zone surrounding the whitepulp areas and in the adjacent red pulp. Transgenic mouse #6 had a fewscattered Russell bodies (FIG. 10F, inset). The red pulp had mild tomoderate extramedullary hematopoiesis, which was comparable to that seenin the control mice (FIG. 10E).

The small intestinal Peyer's patches were mildly to markedly enlarged inthe B7RP1-Fc transgenic mice over those of the control mice (FIG. 10G)and had very large follicles with prominent germinal centers,particularly in transgenic mouse #40 and #32 (FIG. 10H). In addition,there was a minimal (in #32) to mild (in #8 and #33) increase in thenumbers of lymphocytes and plasma cells (admixed with a mild eosinophilinfiltrate in the ileum of mouse #32) in the thickened lamina proprialayer of the mucosa, which was present in the small intestine, but moreprominent in the colon of the transgenic mice. The large intestinallymphoid aggregates (GALT) were also slightly more prominent in someB7RP1-Fc transgenic mice (particularly mouse #8 and #2) than in thecontrol group.

Generally, the other tissues examined, including the thymus, bonemarrow, liver, lung, heart, pancreas, kidneys, adrenal gland, thyroid,parathyroid, trachea, reproductive organs, urinary bladder, mammarygland, skin, skeletal muscle, peripheral nerve, brain, esophagus,stomach, small and large intestine, bone (femur/tibia), stifle joint,white and brown fat appeared normal and comparable to the backgroundchanges detected in the control mice.

The data from this study demonstrate that overexpression of theB7-related protein Fc chimera (B7RP1-Fc) in transgenic mice induces aphenotype characterized by prominent reactive lymphoid hyperplasiadetected in the spleen, peripheral and internal lymph nodes, andgut-associated lymphoid tissue, as follicular hyperplasia, expansion ofT-cell areas and conspicuous plasmacytosis accompanied byhyperglobulinemia in some animals. The plasmacytosis is accompanied byhigher levels of circulating IgG (mean±SD=597±298 mg/ml in transgenicmice vs. 209±80 mg/ml in control littermates, n=7, P<0.05, t test), inparticular IgG2a (217±100 mg/ml vs. 75±29 mg/ml, n=7, P<0.01, t test).The induction of IgG2a is normally associated with a Th1 cytokines suchas IFN-g Thus, B7RP1 induces B- and T-cell proliferation and stimulatesB-cells to differentiate into plasma cells and to produceimmunoglobulin.

These changes are consistent with a persistent systemic immune responsewith hyperstimulation of the humoral arm of the immune system whichresults in B cell stimulation, proliferation, and differentiation toantibody-producing plasma cells throughout the lymphoid organs examined.

We conclude from the marked lymphoid hyperplasia demonstrated in theB7RP1-Fc transgenic mice that B7RP1 protein has significant in vivobiological activity, related to immune system stimulation.

Example 14 Cloning of Human B7RP1

Normal human circulating peripheral lymphocytes were separated from redblood cells using Lymphocyte Separation Medium (ICN Pharmaceuticals).The T-cells were then activated with 10 μg/ml plate bound anti-CD3antibody (Immunotech, Westbrook, Me.), 10 ng/ml PMA, and 500 ng/mlionomycin overnight (16 hours) at 37° C. and 5% CO₂. Total RNA was thenprepared from the cells using TRIzol reagent (Gibco BRL). The cells werepelleted by centrifugation and the cell pellet was resuspended in 1 mlTRIzol reagent for each 5×10⁶ cells and incubated at room temperaturefor 5 min. 0.2 ml chloroform per 1 ml original TRIzol reagent was thenadded. The tubes were shaken vigorously by hand for 15 seconds andincubated for 3 minutes at RT and centrifuged at 13,000 rpm for 15 minat 4° C. Following centrifugation, the clear upper aqueous phase whichcontains the RNA was collected and the sample RNA was precipitated bythe addition of isopropyl alcohol. The solution was then incubated at RTfor 10 min, the RNA pelleted, washed with 75% ethanol, and thencentrifuged at 15,000 rpm for 5 min at 4° C. The pellet was air dried,resuspend in RNAse-free water, then aliquoted, and stored at −80° C.until later use.

The library was constructed using the SuperScript Plasmid System forcDNA Synthesis and Plasmid Cloning (Gibco BRL). Briefly, cDNA insertswith an average size of 2 kb, were ligated into the pSport vector atSalI/Not1 cloning site. The ligated plasmids were electroporated intoElectromax transformation competent E. coli (Gibco BRL), titered andplated at fifteen thousand colonies per LB plate (ampicillin 100 μg/ml).300,000 colonies were lifted onto colony/plaque screen hybridizationtransfer membranes (NEN Life Sciences), denatured in 0.5 N NaOH, 1.5 MNaCl for 5 minutes, then neutralized successively for 5 minutes each inthe following buffers, 1 M Tris HCl pH 8.0, 0.5 M Tris HCl pH 8.0 and1.5 M NaCl and 2×SSC. The filters were then crosslinked by ultravioletirradiation and baked for 30 min at 80° C. in a vacuum oven. The filterswere pre-washed extensively in 2×SSC at 42° C. to remove debris, thenprehybridized at 42° C. in 50% formamide, 5×SSPE, 5×Denhardt's solution,0.5% SDS, 100 μg/ml salmon sperm DNA, for 2 hours.

The human lymphocyte cDNA library was screened with an 895 bp DNAfragment having nucleotides 1-711 as shown in FIG. 3A, 167 bpsimmediately 5′ to the initiator methionize codon in FIGS. 3A and 17 bpsimmediately 3′ to position 711 in FIG. 3A. This upstream 5′ sequence of167 base pairs was obtained by 5′ RACE of the HuB7RP1 cDNA (Example 4)and was released from a TOPO TA vector (Invitrogen, Carlsbad, Calif.) atthe Eco RI restriction enzyme cleavage site. This insert was twicepurified on a 0.8% agarose TAE gel. A DNA gel purification kit (Qiagen)was used to isolate the DNA insert from the agarose.

125 ng of the DNA fragment was labeled with ³²P dCTP (Amersham)following the Redi-Prime 2 (Amersham) random prime labeling systemprotocol. The colony lift filters were then allowed to hybridize withthe probe at 42° C. In the following buffer overnight at 42° C.; 50%formamide, 5×SSPE, 2×Denhardt's solution, 0.5% SDS, 100 mg/ml ssDNA. Thespecific activity of the probe was 2.38×10⁹ cpm/μg DNA, in approximately2 ng labeled probe per ml hybridization buffer. The probe was removedand saved for the next round of screening. The filters were then washedin 2×SSC, 0.1% SDS RT for 15 min, followed by 1×SSC, 0.1% SDS at 55° C.for 15 min, and 1×SSC, 0.1% SDS at 60° C. for 10 min. The filters werewrapped in plastic and exposed to autoradiography film overnight at −80°C. with 2 enhancing screens. Three independent positive clones wereidentified. Exposures were aligned to the bacterial plates and thepositive clones scraped, diluted and replated on LB plates withampicillin 100 μg/ml, grown overnight as before and the colonies werelifted, prepared, and probed as described previously. Three independentclone colonies were isolated, the DNA was isolated, and DNA sequencedfor each clone in triplicate.

The full-length of the human B7RP1 protein is 302 amino acids. Thepolypeptide length and relative position of the transmembrane domain, isconsistent with other B7 family members. The human B7RP1 gene has 43%amino acid identity with the mouse clone. This degree of homology issignificant since the mouse and human CD80 proteins are only 41%identical. Notably conserved between the mouse and human genes are thecysteine residues at amino acid positions 37, 113, 158, 215, and 216.

Example 15 Cloning of Human CRP1

A Genbank blast homology search (GCG, University of Wisconsin) usingmurine B7RP1 sequence (see FIG. 2) retrieved a genomic clone (Gen BankAssession NO. AQ022676) containing a 104 bp sequence that showed highhomology with the murine CRP1 gene. PCR cloning primers were designed tooverlap this sequence.

5′-GCA TAT TTA TGA ATC CCA-3′ (SEQ ID NO: 34)5′-ACT ATT AGG GTC ATG CAC-3′ (SEQ ID NO: 35)

Using the above primers, δ 151 bp DNA fragment of the murine CRP1 wasPCR amplified using the murine CRP1 plasmid described in FIG. 1 andExample 1 as template. 125 ng of the DNA was labeled with 32P dCTP(Amersham) following the Redi-Prime 2 (Amersham) random prime labelingsystem protocol. The colony lift filters from human peripheral bloodlibraries described in Example 15 were then allowed to hybridize withthe probe in the following hybridization buffer overnight (15 hr) at 41°C., 50% formamide, 5×SSPE, 2×Denhardt's solution, 0.5% SDS, 100 μg/mlssDNA. The specific activity of the probe was 3.52×10⁹ cpm/μg DNA, 1.5ng labeled probe/ml hybridization buffer. The probe was pulled off andsaved for the next round of screening. The filters were then washed in2×SSC, 0.1% SDS at RT for 10 min, followed by 1×SSC, 0.1% SDS at 37° C.for 7 minutes, 40° C. for 7 minutes, 44° C. for 7 minutes, then 50° C.for 7 minutes, continually monitoring the rate at which the filters werereleasing the labeled probe. The filters were wrapped in plastic andexposed to film overnight at −80° C. with 2 enhancing screens. Thismethod revealed 9 possible independent positive clones. Exposures werealigned to the bacterial plates and the positive clones scraped,deposited into 200 μl SOC, 2 serial dilutions of 1:10 were performed and70 μl from the second dilution was replated on LB plates containingampicillin at 100 μg/ml and grown overnight as before. The colonies werelifted, prepared and probed as before. Eight independent clones wereisolated and DNA prepared by the Qiagen miniprep method.

A cDNA clone containing an open reading frame of 199 amino acids wasobtained (FIG. 13A). This cDNA clone contained nucleotide and amino acidhomologies to the murine CRP1 clone described in Example 1 and FIG. 1.The nucleotides corresponding to the open reading frame of this humanclone was 77% identical to the murine CRP1 gene. Translation of thehuman sequence and subsequent comparison with the murine CRP1 proteinrevealed 69% amino acid identity with the murine protein (FIG. 13B). Inaddition, the motif between amino acids 114 to 119, “FDPPPF”, wasconserved between the murine and human CRP1 genes. This motifcorresponds to the “MYPPPY” motif in murine and human CD28 that isessential for B7 protein interaction. Furthermore, the cysteines atamino acid positions 42, 109, and 141 are also conserved. Thesecysteines correspond to cysteines in CD28 and CTLA-4 at are involved inIg loop formation and intermolecular disulfide dimerization. The closesimilarity with murine CRP1, and structural similarities with the CD28homology family, indicate that this is the human CRP1 homolog.

Example 16 CRP1 is Expressed on Resting Memory T-lymphocytes

In order to study CRP1 expression on memory T-cells, splenic T-cellswere collected from 6-7 month old mice. These cells were double-stainedusing B7RP1-Fc labeled by an FITC-conjugated anti-human Fc antibody anda PE-conjugated antibody to either CD44, CD45RB, or CD69. Staining withthe B7RP1-Fc fusion protein detects expression of CRP1 protein on theseT-cells. Older mice show more CRP1+ splenic T-cells than younger mice.Interestingly, a conspicuous number of these cells are CD44 high (FIG.14 a) and CD45RB low (FIG. 14 b), a profile typical of memory T-cells.These CRP1+ memory T-cells are in a resting state, since they do notexpress the activation marker CD69 (FIG. 14 c). The expression of CRP1on memory T-cells indicates that CRP1 has costimulatory functions onmemory T-cells.

Example 17 In vitro T-Cell Costimulation Inhibited by Antibodies toB7RP1

To determine if the B7RP1 protein has functional relevance to T-cells,we incubated CD3+ T-cells with the B7RP1-Fc fusion protein and ananti-CD3 antibody in an in vitro proliferation assay. Rabbit anti-mouseB7RP1 polyclonal antibodies or rat anti-mouse B7RP1 monoclonalantibodies were then used to specifically inhibit B7RP1-Fc costimulatedproliferation in vitro.

B7RP1 Rabbit Polyclonal Antiserum Preparation

Three New Zealand white rabbits (5-8 lbs. initial weight) were injectedIM with murine B7RP1 protein. Each rabbit was immunized on day 1 with150 μg of murine B7RP1 protein emulsified in an equal volume of HuntersTiter Max complete adjuvant. Further boosts (days 14 and 28) wereperformed by the same procedure. Antibody titers were monitored by EIA.After the second boost, the antisera revealed moderate antibody titers.A 30 ml production bleed was then obtained from each animal. This wasrepeated each week for 6 weeks. Polyclonal antibodies were then purifiedby protein-A agarose chromatography, followed by negative selection Fcprotein affinity chromatography and positive selection by B7RP1-Fcaffinity chromatography.

Rat Anti-Murine B7RP1 Monoclonal Antibody Preparation

Rat anti-murine B7RP1 monoclonal antibodies were generated as describedin Practical Immunology, second edition (1980; L. Hudson and F. C. Hay;Blackwell Scientific Publications; St. Louis, Mo.). Briefly, Lou rats(Harlan; Indianapolis, Ind.) were injected intraperitoneally withmuB7RP1-Fc fusion protein emulsified in Freund's Adjuvant at 4 weekintervals. Three days prior to fusion, rats were boosted intravenouslywith soluble muB7RP1. On the day of fusion, the animal was sacrificedunder carbon dioxide and the spleen removed aseptically. Single cellsuspension was generated using a tissue stomacher. Both splenocytes andY3-Ag1.2.3 myeloma cells (American Type Culture Collection; Rockville,Md.) were washed in serum-free media then fused by the addition ofpolyethylene glycol (PEG 1500; Boehringer Mannheim Biochemicals;Indianapolis, Ind.). The cells were rinsed once, resuspended inserum-containing media, and plated into 96-well tissue culture plates.Ten to 12 days later, media from each well was tested for specificantibody to B7RP1 via a direct Enzyme-linked Immunosorbent Assay (EIA).Cells from wells indicating potential binding were grown to 10 mlcultures and frozen in liquid nitrogen. Media from each culture wasfurther tested in flow cytometry and in a functional T-cellproliferation assay. Those determined to be of interest by these methodswere plated to single cell colonies, selected again by EIA, and finalcell lines maintained for antibody generation. Antibodies were purifiedfrom the cell media by protein A agarose chromatography.

T-cell Preparation and T-cell Proliferation Assay

T-cells from the spleens of C57Bl/6 mice (8-12 week-old females, CharlesRiver Laboratories) were purified by negative selection through a murineT-cell enrichment column (R&D Systems). The T-cells were then eitherused directly or further purified by antibody and complement lysis asfollows. Cells were resuspended (2.5×10⁶ cells/ml) in RPMI mediumcontaining antibodies (all at 10 μg/ml and from Pharmingen) againstmurine CD11b (Clone M1/70), NK-1.1 (Clone PK136), CD8a (Clone 53-6.7),I-A^(b) (Clone M5/114.15.2), CD11c (Clone HL3), and the B220 antigen(Clone RA3-6B2). The cells were then incubated on ice for 30 min,pelleted at 1200 rpm, resuspended in 4:1 vol/vol of RPMI: rabbitcomplement (Sigma, #S-7764), and incubated for an additional 30 min at37° C. The cells were pelleted again, and the complement treatment wasrepeated. Before plating, the cells were washed with RPMI containing 10%FCS. U-bottomed 96 well plates were coated with an anti-CD3 antibody(Clone 145-2C11, Pharmingen) at concentrations ranging between 0 and 1.2μg/ml), and anti-human IgG Fab₂ (Sigma, 12.5 μg/ml) overnight at 4° C.,followed by a 6-9 hr incubation at 37° C. T-cells (1×10⁵/well) werecultured in the absence or presence of various Fc fusion proteins for 48hr and were pulsed during the last 18 hours with 1 μCi of ³H-thymidine.Control Fc proteins included a fusion protein of OPG and Fc and anonfused Fc protein fragment. The cells were then harvested and theincorporated radioactivity was counted. B7RP1-Fc co-stimulates T-cellsto proliferate in a dose-dependent fashion (FIG. 15 a), and an anti-B7RP1-Fc antibody specifically inhibits this co-stimulationdose-dependently (FIG. 15 b).

Example 18 Inhibitors of the CRP1/B7RP1 Pathway Decrease the Onset ofRheumatoid Arthritis Induced by Collagen

Collagen-induced arthritis (CIA) is an animal model of autoimmunepolyarthritis in rodents and primates that has many similarities withrheumatoid arthritis in humans. Immunization with heterologous speciesof type II collagen (CII) induces an autoimmune response to CII thatleads to the development of CIA in susceptible mouse strains. Congenicstrains of mice with H-2^(r) and H-2^(q) are highly susceptible to CIA.CIA is mediated by the synergistic effects of both CII-reactive T-cellsand antibodies. Porcine CII (Nabozny et al., Autoimmunity 20, 51-58(1995)) was dissolved in 0.01N acetic acid at a concentration of 2 mg/mland then was emulsified at a 1:1 ratio with CFA (Difco). Arthritissusceptible B10.RIII (H-2^(r)) mice (Jackson Laboratories, Bar Harbor,Me.) were immunized with 100 μl of emulsion intradermally at the base ofthe tail. Mice were monitored 2-3 times per week for the development ofarthritis. Arthritis severity was determined using a grading system foreach paw as follows: 0: no arthritis; 1: redness or swelling in 1-3toes; 2: severe swelling of paw; 3: joint ankylosis. The score of eachlimb was summed to give a severity range from 0 to 12 for each animal.

Mice were injected with 100 ug (in 200 μL) of protein intraperitoneallytwice per week. The treatment was begun 1 day after immunization withporcine CII and was stopped at day 52 post-immunization. The experimentwas conducted in treatment groups of 10 mice, and animals with scores of1 or above were scored as positive. The results are shown in FIG. 16 andTable 1.

TABLE 1 Effect of CRP1, B7RP1, CTLA-4, and B7.2 Fc fusion proteins onthe onset of arthritis Treatment Mean +/− s.d. groups day of onsetCTLA4-Fc 60.0 ± 0.0 CRP1-Fc 48.9 ± 13.2 B7.2-Fc 28.4 ± 14.1 B7RP1-Fc33.9 ± 16.6 PBS 37.7 ± 17.1

In mice treated with CRP1-Fc fusion protein, the onset of arthriticsymptoms were delayed by approximately 10 days as compared to the PBStreated mice. This demonstrates that the inhibition of the CRP1/B7RP1pathway can alleviate disease symptoms in this mouse model of rheumatoidarthritis.

Mice treated with B7RP1-Fc or B7.2-Fc showed an earlier onset of disease(Table 1 and FIG. 16 a) with an increase in arthritic severity (FIG. 16b) as compared to the PBS-treated controls. This indicates that theB7RP1-Fc fusion protein enhances the T-cell immune response. Suchactivity may be useful in generating anti-tumor immunity in vivo.

The opposite effects by CRP1-Fc and B7RP1-Fc in this mouse model ofrheumatoid arthritis indicate that the pathway can be manipulated toeither enhance or inhibit the disease progression. Targeting the CRP1protein with soluble B7RP1-Fc enhances the disease, while theinteraction of soluble CRP1-Fc with B7RP1 inhibits the disease symptoms.

Example 19 B7RP1-Fc Induces an Inflammatory Bowel Disease Phenotype inTransgenic Mice

Persistent overexpression of the B7-related protein (B7RP1-Fc) in22-to-25-week-old transgenic mice (Example 12) induces a strikingphenotype of inflammatory bowel disease (IBD) with marked thickening andchronic inflammation of the small and large bowels (enterocolitis) andweight loss in some animals. Histologically, the most severeinflammatory changes were found in the proximal and distal colon, withmilder changes in the small intestine. The proximal colon was markedlythickened with fissuring ulceration, transmural inflammation, andhypertrophy of the colonic mucosa, while the distal colon had diffusemucosal hypertrophy (or focal erosion and glandular atrophy) withoutulceration. The proximal small intestine had mild to marked mucosalhypertrophy with milder inflammatory changes, while the distal smallintestine (ileum) had mild mucosal hypertrophy in some animals andatrophy in other mice. The intestinal changes were most severe andconsistently found in the female B7RP1 transgenic mice, but were alsoobserved in several of the male transgenic mice in this study.

It is interesting to note that the histologic features found in theproximal colon, including the fissuring ulceration and the transmuralchronic granulomatous inflammation with multinucleated giant cells, moreclosely resemble those seen in Crohn's disease than ulcerative colitisin humans. Morphologically, this colitis also mimics the IBD describedin mice deficient in interleukin-10, which develop wasting, anemia, andenterocolitis affecting their entire intestinal tract (Kuhn et al. 1993;Sartor 1995; Leach et al. 1999). As in the IL-10 knockout mice, theinitial changes in the B7RP1-Fc transgenic mice consist of mild, focalinfiltrates of inflammatory cells in the lamina propria without colonicepithelial hyperplasia (Example 13). In older mice, the affected colonicsegments become thickened due to glandular hypertrophy/hyperplasia andchronic inflammation. The proximal and distal colons of the B7RP1-Fcmice had moderate to severe colitis with histologic features ofinflammatory bowel disease (IBD). The affected segments of the proximalcolon (FIG. 17B-17D) were diffusely thickened, due to prominentglandular epithelial hypertrophy and hyperplasia with elongation anddilatation of the mucosal glands (FIG. 17B), which had increased numbersof mitotic figures and rare crypt abscesses, but retained goblet cellswith mucin (FIG. 17D). The mucosa had diffuse chronic inflammation inthe lamina propria, which in some animals extended transmurally toinvolve the underlying layers of the gut wall, including the submucosa,muscularis, serosa, and the adjacent mesenteric fat tissue (FIG.17B-17C). The inflammatory infiltrates consisted of lymphocytes(predominantly CD3+, CD44+ T-cells), plasma cells, and epithelioidmacrophages (FIG. 17F) mixed with some neutrophils and occasionalmultinucleated giant cells (FIG. 17E), characteristic of chronicgranulomatous inflammation. Lymphoid aggregates (mostly B220+ cellsmixed with small numbers of CD3+ cells) were also present in the mucosaand around smaller blood vessels in the submucosa and deeper layers,including mesenteric fat (FIG. 17C). The lumen contained mucopurulent ormucous exudate (FIG. 17D). Severe evidence of colitis, with multifocalfissuring ulceration of the mucosa and transmural inflammation (FIG.17B-17C), was found in these B7RP1-Fc transgenic mice.

The distal colon of the B7RP1-Fc transgenic mice was also diffuselythickened and hyperplastic with elongation, basophilia, and dilatationof the colonic glands (FIG. 18B-18G), some of which contained cryptabscesses (FIGS. 18D and 18F) and mucus. The lamina propria had a milddiffuse inflammatory infiltrate of lymphocytes (predominantly CD3+,CD44+ cells, particularly in the superficial mucosa; FIG. 18E), as wellas plasma cells and focal aggregates of epithelioid macrophages mixedwith some neutrophils. Lymphoid aggregates (of predominantly B220+cells; FIGS. 18D and 18F) were also scattered throughout the mucosa. Thesmall intestine of B7RP1-Fc transgenic mice had more variable changes,including mild to focally marked mucosal and crypt hypertrophy andhyperplasia (FIGS. 19B and 19D with crypt/villus ratios ranging from 1:4to 1.5:1, as compared to 1:10 in the control mice) accompanied by apredominantly lymphoplasmacytic infiltrate in the lamina propria. Themucosal hyperplasia was most prominent in the proximal small intestine,including the duodenum (FIG. 19B) and particularly the jejunum (FIG.19D). The crypt architecture was focally deranged and dysplastic in themost severely affected mice (FIG. 19D). In contrast, the distal smallintestines (ileum) of some mice, had mild, patchy villous atrophy of theileal mucosa (FIG. 19F) with blunting, thickening or focal loss of villi(with a crypt:villus ratio of 1:1 or less, instead of the normal ratioof 1:2), while other mice had mild ileal mucosal hypertrophy.

The B7RP1-Fc fusion protein acts to activate cells that are responsiblefor eliciting a phenotype very similar to that of human Crohn's disease.This indicates that the cells that may be responsible for theinflammation in Crohn's disease are activated by the B7RP1-Fc fusionprotein. Soluble protein, antibody or small molecule inhibitors of B7RP1may therefore be useful in inhibiting IBD.

Example 20 The B7RP1-Fc Fusion Protein Inhibits Tumor Growth in Mice

To examine the effect of B7RP1 and CRP1 on the growth of the immunogenicmurine Meth A sarcoma, we investigated whether the soluble B7RP1-Fcaffects the growth of an established Meth A sarcoma in Balb/c mice.

Exponentially growing Meth A sarcoma cells were implanted by intradermalinjection of 0.5 million cells in the abdomen of Balb/c mice on day 0.On day 7, when the tumors reached ˜100 mm³, the mice were treated witheither vehicle (PBS) or B7RP1-Fc (8 mg/kg), subcutaneously in the neckon days 7, 10, 14, and 17. The bidimensional diameters of the tumorswere measured by calipers and the tumor volume (in mm³) was estimatedusing the formula: Tumor volume=[{(width)2×length}/2]. The tumor growthwas monitored up to day 28. Each group had eight mice.

The Meth A sarcoma growth pattern of the control tumor was bi-phasic: aslow initial phase was followed by a relatively rapid exponential phase.In B7RP1-Fc treated mice, the growth of the tumor was significantlyslower in the rapid exponential phase. On day 28, the average volumes ofthe control and B7RP1-Fc treated mice were 1410 mm³ and 580 mm³,respectively (FIG. 20). Therefore, B7RP1-Fc treatment inhibited tumorgrowth significantly in this model. The data strongly suggest thebeneficial therapeutic utility of the soluble B7RP1-Fc protein, andother activators of the B7RP1/CRP1 pathway, in the treatment ofimmunogenic tumors.

Immunologic anti-tumor activity is closely associated with cytolyticT-lymphocyte (CTL) function. Consistently, the B7RP1-Fc protein isexpressed on cytolytic CD8+ T-cells (Example 9, FIG. 6). These datastrongly support B7RP1 functions on cytolytic CD8+ T-cells. B7RP1-Fc, orother stimulators of the B7RP1/CRP1 pathway, may therefore be used toenhance cytolytic T-cell and cellular immune functions for a number ofnon-cancer-related indications.

Example 21 Inhibition of Human B7RP1 Activity in vitro

To determine if human B7RP1 has positive co-stimulatory properties, wetested cells expressing human B7RP1 and human B7RP1-Fc fusion protein inT-cell proliferation assays. The human B7RP1-Fc fusion protein wasconstructed by fusing gene sequences corresponding to amino acids 1 to247 to a partial human IgG1 gene sequence (Example 14). The humanCRP1-Fc fusion protein was constructed by fusing gene sequencescorresponding to amino acids 1 to 146 to a partial human IgG1 genesequence (Example 2). The methods of construction, expression andpurification of both fusion proteins were conducted as described inExample 7. B7RP1-Fc demonstrated co-stimulatory activities that aredependent on anti-CD3 stimulation (FIG. 21 a). In addition, thisactivity can be specifically inhibited with soluble CRP1-Fc protein(FIG. 21 b). Similar co-stimulatory effects were obtained using CHOcells that express membrane-bound, human B7RP1, containing the entirecoding sequence (FIG. 21 c).

The production of cytokines by human T-cells under the above in vitroproliferation conditions was determined. Supernatants from T-cellcultures stimulated for 48 and 72 hours were analyzed for IL-2, IL-10,and IFN-gamma by ELISA according to the manufacturer's specifications(BioSource International). The IFN-gamma and IL-10 levels weresignificantly increased; however, unlike the case with CD28co-stimulation, IL-2 was not notably induced (FIG. 21 d). The increasedlevels of IFN-gamma, a Th1 cytokine, correlate with the B7RP1 functionsto increase IgG2a, as described in Example 13.

In vitro T-cell co-stimulation assays were conducted as follows. Highlypurified human T-cells (>98% CD3+) were isolated by negative selectionof fresh or thawed, adherence depleted PBMC using mAb labeled magneticbeads (Miltenyi Biotec). T-cells (1×10⁵ cells/well) were cultured intriplicate wells in 96 well plates in 200 μl/well RPMI+10% FCS. Toevaluate B7RP1-Fc co-stimulation, various concentrations of anti-CD3(Pharmingen) and 10 μg/ml anti-human IgG Fc (Sigma) in 100 μl 1×PBS werepre-coated onto U bottom plates by an incubation at 4° C. overnight. Theunbound anti-CD3 and anti-human IgG Fc were removed, and the cells werecultured in the presence or absence of various concentrations ofB7RP1-Fc, OPG-Fc control or anti-CD28 (Pharmingen). For CRP1-Fcinhibition of B7RP1-Fc co-stimulation, T-cells were cultured in 0.33μg/ml anti-CD3 and 10 μg/ml anti-human IgG Fc pre-coated wells with 0.5μg/ml B7RP1-Fc in the presence of serially diluted CRP1-Fc or OPG-Fc,starting at 10 μg/ml. To evaluate co-stimulation by CHO cells expressingB7RP1, T-cells were cultured in flat bottom plates with variousconcentrations of soluble anti-CD3 in the presence or absence of variousamounts of mitomycin-C treated CHO B7RP1 cells or CHO vector cells. Totest for T-cell proliferation, cultures were pulsed with 1 uCi/well[³H]TdR during the last 18 hrs of a 72 hr culture. T-cell proliferationwas determined by [³H]TdR incorporation. The results of onerepresentative experiment from three random donors are expressed as meanCPM incorporated +/−SD. For analyses of cytokine production, cells werecultured for 48 and 72 hours and supernatants were collected for ELISA.

These experiments show that the extracellular portion of human B7RP1, asdescribed in Example 14, when fused to a human Fc fragment, canco-stimulate T-cells in vitro. This co-stimulation is inhibited byCRP1-Fc and thus demonstrates how a soluble inhibitor of human B7RP1 mayfunction. In vitro assays, such as that described here using human B7RP1and CRP1, could be used to screen for antibody, soluble protein,peptibody, or small molecule inhibitors of B7RP1/CRP1 activity.

Example 22 B7RP1 Transgenic Mice have Increased IgE Levels

Transgenic mice described in Example 12 were analyzed for serum IgEantibodies. Orbital sera from B7RP1-Fc transgenic mice and littermatecontrols were analyzed for IgE by an ELISA protocol. 96-well polystyreneEIA plates (Coster, Cambridge, Mass.) were coated with 50 μl of a 5μg/ml solution of rat anti-mouse IgE antibody (BD Pharmingen, San Diego,Calif.) in 50 mM Na₂CO₃/NaHCO₃ buffer (pH9.6 at room temperature for 2hr then at 4° C. overnight. The subsequent procedures were conducted atroom temperature. After blocking with assay diluent (1% BSA, 1% normalgoat serum, and 0.2% tween-20 in PBS) for 30 min, serum samples andmouse IgE standards (BD Pharmingen) were added into the wells, and theplates were incubated for 2 hr. The wells were washed 3 times with KPLwash buffer (KPL, Gaithersburg, Md.). Biotin-conjugated goat anti-ratantibody (BD Pharmingen) was added and incubated for 1 hr. After washing3 times with KPL wash buffer, horseradish peroxidase-conjugatedstreptavidin (BD Pharmingen) was added to the wells, and the plates wereincubated for 30 min. After the wells were washed 5 times with KPL washbuffer, TMB-hydrogen peroxide substrate solution (KPL) was added, andthe plates were incubated for 10 min. The reaction was stopped by theaddition of 1M phosphoric acid, and the absorbance was read at awavelength of 450 nm. The IgE levels in the sera from the B7RP1-Fctransgenic mice were approximately three times higher than those in thelittermate controls (FIG. 22). This increase in the IgE serum levelsindicates that B7RP1 regulates IgE expression.

Example 23 IgE-Mediated Response of CRP1 Knockout Mice

The CRP1 gene in mice was disrupted by deleting a genomic fragmentcorresponding to nucleotides 318-591 of the murine CRP1 cDNA sequence(see SEQ ID NO: 1). The murine CRP1 gene was isolated from a 129Jlibrary using the full-length (800 bp) cDNA probe (Yoshinaga et al.Nature 402, 827-832 (1999). The targeting vector, which replaced a 2.8kb genomic fragment with a neomycin resistance (neo) cassette in senseorientation relative to CRP1 transcription, was electroporated into E14embryonic stem (ES) cells (129/Ola, available from the American TypeCulture Collection, Manassas, Va. under accession no. CRL-1821). AfterG418 selection, homologous recombinants were identified by PCR using theprimer pair GAG ACT CAT GCT GTG GTT TCA GG (SEQ ID NO: 38) and TTC GCCAAT GAC AAG ACG CTG G (SEQ ID NO: 39) and verified by Southern blotting.Chimeric mice generated from CRP1^(+/−) ES clones were crossed withC57BL6 females to produce CRP1^(+/−) mice. Germline transmission of theCRP1 mutation was assessed by PCR and Southern blot analysis of tailDNA. CRP1^(+/−) mice generated by the intercrossing of heterozygousoffspring were born at the expected Mendelian frequency and were viable,fertile and of normal size. To verify that the CRP1 mutation hadabolished CRP1 expression, activated T-cells from CRP1^(+/−) mice andcontrol littermates were analyzed by flow cytometry. Upon in vitroT-cell activation, CRP1 was expressed on the surface of both CD4⁺ andCD8³⁺ wild type T-cells, but was undetectable on CRP1^(−/−) T-cells.

To investigate the role of CRP1 in T-cell mediated B cell antibodyproduction and isotype class switching, CRP1^(−/−) mice and littermatecontrols were immunized with 200 μg of nitrophenol conjugated ovalbumin(NP-OVA; Biosearch Technologies, Inc.) adsorbed to alumintraperitoneally. Blood was collected from mouse tails every weekfollowing immunization and the levels of nitrophenol (NP)-specific IgMand IgG₁ as well as the levels of OVA-specific IgE were assessed in theserum.

Titers of NP-specific IgG₁ and IgM antibodies were assessed usingsandwich ELISA (Southern Biotechnology Associates) in which ELISA plateswere coated with NP (23)-bovine serum albumin at 50 μg/ml in PBS orcarbonate buffer pH 9.2 (Sigma). The arbitrary values obtained by usingthe SOFTmaxPRO (Molecular Devices) ELISA analysis program were based onthe titration curve of the isotype standard used on each plate. Levelsof ovalbumin-specific IgE were detected using an antigen-capture(biotinylated ovalbumin) ELISA method as previously described (Stampfliet al. Am J. Respir. Cell Mol. Biol. 21, 317-326 (1999)). The ELISA wasstandardized using serum obtained from mice sensitized to ovalbuminaccording to a conventional intraperitoneal sensitization model(Ohkawara et al. Am. J. Respir. Cell Mol. Biol. 16, 510 (1997)).

The levels of NP-specific IgM were normal (day 7); however, asignificant reduction in the levels of NP-specific IgG₁ at day 14 and 21post-immunization, indicating that T-cell-dependent antibody productionwas severely impaired in CRP1^(−/−) mice. Furthermore, we observed thatOVA-specific IgE were completely undetectable in the serum of ICOS^(−/−)mice at day 21 and 28 post-immunization, whereas isotype switchingoccurred in both heterozygous and wild type control mice, asdemonstrated by the presence of IgE in the serum of these animals. (seeTable 3). These findings indicate that ICOS plays a role of primaryimportance in controlling T-B cell collaboration and switching of IgG₁and IgE isotype classes.

TABLE 3 IgE isotype switching in CRP1−/− mice Days post- immunizationCRP1^(+/+) CRP1^(+/−) CRP1^(−/−) Day 21 4/8 5/9 0/7 Day 28 4/6 8/9 0/8

Example 24 Effects of B7RP1 and B7.2 in a Contact Hypersensitivity Model

In order to induce contact hypersensitivity, female Balb/c mice (9 to 14weeks old; Charles River Laboratories, Wilmington, Mass.) were firstsensitized by applying a 1% solution of oxazolone (Sigma, St. Louis,Mo.) in acetone and olive oil onto the shaved skin of the abdomen. Sevendays after sensitization, mice were challenged (day 0) by applying theoxazolone solution onto the right ear. The acetone and olive oil solventwas applied at the same time onto the left ear as a control. Thethickness of the ears was measured daily with a micrometer (Mitutoyo,Kawasaki, Japan) starting immediately before challenge. The differencein ear thickness (ΔET) between right and left ear was used to expressthe results (McHale et al. J. Immunol. 162, 1648 (1999)).

It has been previously observed that administration of B7RP1-Fc fusionprotein in this model exacerbates contact hypersensitivity both at thetime of sensitization (induction of the primary immune response) or atthe time of challenge (induction of the secondary immune response),although the effects are more pronounced when administration occurs atthe time of challenge (Yoshinaga et al. Nature 402, 827-832 (1999)).

In order to study the combined effects of B7RP1-Fc and B7.2-Fc oncontact hypersensitivity, B7RP1-Fc and B7.2-Fc were given at twodifferent doses either alone or in combination around the time ofchallenge. At high dose (2 mg/Kg of B7RP1-Fc alone, 2 mg/Kg of B7.2-Fcalone, and 1 mg/Kg of each in combination), B7RP1-Fc, B7.2-Fc, andB7RP1-Fc+B7.2-Fc increased ΔET compared to Fc (2 mg/Kg) from day 3 (FIG.23A). At this dose B7RP1-Fc and B7RP1-Fc+B7.2-Fc increased ΔET more thanB7.2-Fc from day 4 (FIG. 23A). B7RP1-Fc+B7.2-Fc also increased ΔET morethan B7RP1-Fc from day 4 (FIG. 23A). At low dose (0.4 mg/Kg of B7RP1-Fcalone, 0.4 mg/Kg of B7.2-Fc alone, and 0.2 mg/Kg of each incombination), B7RP1-Fc and B7RP1-Fc+B7.2-Fc increased ΔET compared to Fc(0.4 mg/Kg) from day 4, while B7.2-Fc did not significantly change ΔET(FIG. 23B). Also at this dose B7RP1-Fc and B7RP1-Fc+B7.2-Fc increasedΔET more than B7.2-Fc, and B7RP1-Fc+B7.2-Fc more than B7RP1-Fc (FIG.23B). Thus, half of full doses of B7RP1-Fc combined with half of fulldoses of B7.2-Fc increased ΔET on days 4 to 7 more than full doses ofB7RP1-Fc alone or of B7.2 alone, indicating that B7RP1-Fc and B7.2-Fcsynergistically interacted.

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed.

1. A method of decreasing IgE production comprising administering aB7RP1 antagonist in an amount effective to decrease IgE production,wherein the B7RP1 antagonist is an antibody which binds B7RP1 andpartially or completely inhibits IgE production.
 2. A method ofpreventing or treating an IgE-mediated disorder comprising administeringa therapeutically effective amount of a B7RP1 antagonist, wherein theB7RP1 antagonist is an antibody which binds B7RP1 and partially orcompletely inhibits IgE production.
 3. The method of claim 2 wherein theIgE-mediated disorder is asthma or an allergic disorder.
 4. The methodof claim 1 or 2 further comprising administering an IgE antagonist. 5.The method of claim 4 wherein the IgE antagonist is an anti-IgEantibody.